Anti-PD-L1 Antibodies

ABSTRACT

Anti-PD-L1 antibodies are disclosed. Also disclosed are pharmaceutical compositions comprising such antibodies, and methods of using such antibodies to restore T-cell function in T-cells exhibiting T-cell exhaustion or T-cell anergy.

FIELD OF THE INVENTION

The present invention relates to antibodies that bind to programmeddeath-ligand 1 (PD-L1).

BACKGROUND TO THE INVENTION

T-cell exhaustion is a state of T-cell dysfunction that arises duringmany chronic infections and cancer. It is defined by poor T-celleffector function, sustained expression of inhibitory receptors and atranscriptional state distinct from that of functional effector ormemory T-cells. Exhaustion prevents optimal control of infection andtumors. (E John Wherry, Nature Immunology 12, 492-499 (2011)).

T-cell exhaustion is characterized by the stepwise and progressive lossof T-cell functions. Exhaustion is well-defined during chroniclymphocytic choriomeningitis virus infection and commonly develops underconditions of antigen-persistence, which occur following many chronicinfections including hepatitis B virus, hepatitis C virus and humanimmunodeficiency virus infections, as well as during tumor metastasis.Exhaustion is not an uniformly disabled setting as a gradation ofphenotypic and functional defects can manifest, and these cells aredistinct from prototypic effector, memory and also anergic T cells.Exhausted T cells most commonly emerge during high-grade chronicinfections, and the levels and duration of antigenic stimulation arecritical determinants of the process. (Yi et al., Immunology April 2010;129(4):474-481).

Circulating human tumor-specific CD8⁺ T cells may be cytotoxic andproduce cytokines in vivo, indicating that self- and tumor-specifichuman CD8⁺ T cells can reach functional competence after potentimmunotherapy such as vaccination with peptide, incomplete Freund'sadjuvant (IFA), and CpG or after adoptive transfer. In contrast toperipheral blood, T-cells infiltrating tumor sites are oftenfunctionally deficient, with abnormally low cytokine production andupregulation of the inhibitory receptors PD-1, CTLA-4, and TIM-3.Functional deficiency is reversible, since T-cells isolated frommelanoma tissue can restore IFN-γ production after short-term in vitroculture. However, it remains to be determined whether this functionalimpairment involves further molecular pathways, possibly resemblingT-cell exhaustion or anergy as defined in animal models. (Baitsch etal., J Clin Invest. 2011; 121(6):2350-2360).

Programmed cell death 1 (PD-1), also called CD279, is a type I membraneprotein encoded in humans by the PDCD1 gene. It has two ligands, PD-L1and PD-L2. PD-L1, also called CD274 or B7 homolog 1 (B7-H1) is a 40 kDatype I transmembrane protein encoded in humans by the CD274 gene.

PD-1 is expressed on the surface of activated T cells, and PD-L1 isexpressed on the surface of antigen presenting cells (APCs), such asdendritic cells and macrophages. PD-L1 is also overexpressed in severaltumors, including breast, lung, bladder, head and neck, and othercancers. When PD-L1 or PD-L2 bind to PD-1, an inhibitory signal istransmitted into the T cell, which reduces cytokine production andsuppresses T-cell proliferation.

The PD-1 pathway is a key immune-inhibitory mediator of T-cellexhaustion. PD-1 functions to limit the activity of already activated Tcells in the periphery during the inflammatory response to infection inorder to limit autoimmunity. Blockade of this pathway can lead to T-cellactivation, expansion, and enhanced effector functions. As such, PD-1negatively regulates T cell responses. PD-1 has been identified as amarker of exhausted T cells in chronic disease states, and blockade ofPD-1:PD-L1 interactions has been shown to partially restore T cellfunction. (Sakuishi et al., JEM Vol. 207, Sep. 27, 2010, pp2187-2194).

Methods and compositions for the treatment of persistent infections andcancer by inhibiting the PD-1 pathway are disclosed in WO 2006/133396.Human monoclonal antibodies to PD-L1 are described in WO 2007/005874,US2011/209230, U.S. Pat. No. 8,217,149 and WO 2014/055897.

SUMMARY OF THE INVENTION

The present invention is concerned with antibodies, or antigen bindingfragments, that bind to PD-L1. Heavy and light chain polypeptides arealso disclosed. The antibodies, antigen binding fragments andpolypeptides may be provided in isolated and/or purified form and may beformulated into compositions suitable for use in research, therapy anddiagnosis.

In some embodiments the antibody, or antigen binding fragment, orpolypeptide may be effective to restore T-cell function in T-cells, e.g.CD8⁺ T-cells, exhibiting T-cell exhaustion or T-cell anergy.

In one aspect of the present invention an antibody, or antigen bindingfragment, is provided, the amino acid sequence of the antibody maycomprise the amino acid sequences i) to iii), or the amino acidsequences iv) to vi), or preferably the amino acid sequences i) to vi):

(SEQ ID NO: 9) i) LC-CDR1: one of SGRSSNIASHDVF,  (SEQ ID NO: 12)GGDNIGRKSVH,  (SEQ ID NO: 15) SGSSSNIGNNYVS,  or  (SEQ ID NO: 18)TGSSSNIGAGYDVH; (SEQ ID NO: 10) ii) LC-CDR2: one of ETNKRPW,(SEQ ID NO: 13) DDGDRPS, (SEQ ID NO: 16) DNNERLS, or  (SEQ ID NO: 19)GNSNRPS; (SEQ ID NO: 11) iii) LC-CDR3: one of GAWDSGLTGML, (SEQ ID NO: 14) QAWDSTVV,  (SEQ ID NO: 17) GTWDSSLSVVV,  or (SEQ ID NO: 20) QSYDSSLSGSYVV; (SEQ ID NO: 21) iv) HC-CDR1: SYAIS;(SEQ ID NO: 22) v) HC-CDR2: RIIPILGIANYAQKFQG; (SEQ ID NO: 26)vi) HC-CDR3: X₁X₂X₃X₄X₅X₆SX₇X₈AFDX₉;or a variant thereof in which one or two or three amino acids in one ormore of the sequences (i) to (vi) are replaced with another amino acid,where X₁=absent (i.e. no amino acid) or G, X₂=G or S, X₃=G or Y, X₄═S, Gor H, X₆═Y or G, X₆=G, N or Y, X₇=L or Y, X₈═Y or G, X₉═I or Y.

In some embodiments HC-CDR3 is one of GGSYGSLYAFDI (SEQ ID NO:23),GGYGGNSLYAFDI (SEQ ID NO: 24), or SGHGYSYGAFDY (SEQ ID NO:25).

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

(SEQ ID NO: 9) LC-CDR1: SGRSSNIASHDVF (SEQ ID NO: 10) LC-CDR2: ETNKRPW(SEQ ID NO: 11) LC-CDR3: GAWDSGLTGML

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

(SEQ ID NO: 12) LC-CDR1: GGDNIGRKSVH (SEQ ID NO: 13) LC-CDR2: DDGDRPS(SEQ ID NO: 14) LC-CDR3: QAWDSTVV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

(SEQ ID NO: 15) LC-CDR1: SGSSSNIGNNYVS (SEQ ID NO: 16) LC-CDR2: DNNERLS(SEQ ID NO: 17) LC-CDR3: GTWDSSLSVVV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

(SEQ ID NO: 18) LC-CDR1: TGSSSNIGAGYDVH (SEQ ID NO: 19) LC-CDR2: GNSNRPS(SEQ ID NO: 20) LC-CDR3: QSYDSSLSGSYVV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

(SEQ ID NO: 21) HC-CDR1: SYAIS (SEQ ID NO: 22)HC-CDR2: RIIPILGIANYAQKFQG (SEQ ID NO: 26)HC-CDR3: X₁X₂X₃X₄X₅X₆SX₇X₈AFDX₉where X₁=absent or G, X₂=G or S, X₃=G or Y, X₄═S, G or H, X₅═Y or G,X₆=G, N or Y, X₇=L or Y, X₈═Y or G, X₉═I or Y.

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

(SEQ ID NO: 21) HC-CDR1: SYAIS (SEQ ID NO: 22)HC-CDR2: RIIPILGIANYAQKFQG (SEQ ID NO: 23) HC-CDR3: GGSYGSLYAFDI

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

(SEQ ID NO: 21) HC-CDR1: SYAIS (SEQ ID NO: 22)HC-CDR2: RIIPILGIANYAQKFQG (SEQ ID NO: 24) HC-CDR3: GGYGGNSLYAFDI

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

(SEQ ID NO: 21) HC-CDR1: SYAIS (SEQ ID NO: 22)HC-CDR2: RIIPILGIANYAQKFQG (SEQ ID NO: 25) HC-CDR3: SGHGYSYGAFDY

The antibody may comprise at least one light chain variable regionincorporating the CDRs shown in FIG. 1 or 3. The antibody may compriseat least one heavy chain variable region incorporating the CDRs shown inFIG. 2 or 3.

The antibody may comprise at least one light chain variable region(V_(L)) comprising the amino acid sequence of one of SEQ ID NOs 1, 9,10, 11; or 2, 12, 13, 14; or 3, 15, 16, 17; or 4, 18, 19, 20, or one ofthe amino acid sequences shown in FIG. 1 or an amino acid sequencehaving at least 70%, more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to one of SEQ ID NOs SEQ ID NOs 1, 9, 10, 11; or2, 12, 13, 14; or 3, 15, 16, 17; or 4, 18, 19, 20, or to the amino acidsequence of the V_(L) chain amino acid sequence shown in FIG. 1.

The antibody may comprise at least one heavy chain variable region(V_(H)) comprising the amino acid sequence of one of SEQ ID NOs 5, 21,22, 23; or 6, 21, 22, 23; or 7, 21, 22, 24; or 8, 21, 22, 25; or 35, 21,22, 25, or one of the amino acid sequences shown in FIG. 2 or an aminoacid sequence having at least 70%, more preferably one of at least 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%, sequence identity to one of SEQ ID NOs 5, 21, 22, 23;or 6, 21, 22, 23; or 7, 21, 22, 24; or 8, 21, 22, 25; or 35, 21, 22, 25,or to the amino acid sequence of the V_(H) chain amino acid sequenceshown in FIG. 2.

The antibody may comprise at least one light chain variable regioncomprising the amino acid sequence of one of SEQ ID NOs 1, 9, 10, 11; or2, 12, 13, 14; or 3, 15, 16, 17; or 4, 18, 19, 20, or one of the aminoacid sequences shown in FIG. 1 (or an amino acid sequence having atleast 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100%, sequence identity to one of SEQ ID NOs 1, 9, 10,11; or 2, 12, 13, 14; or 3, 15, 16, 17; or 4, 18, 19, 20, or to one ofthe amino acid sequences of the V_(L) chain amino acid sequence shown inFIG. 1) and at least one heavy chain variable region comprising theamino acid sequence of one of SEQ ID NOs 5, 21, 22, 23; or 6, 21, 22,23; or 7, 21, 22, 24; or 8, 21, 22, 25; or 35, 21, 22, 25, or one of theamino acid sequence shown in FIG. 2 (or an amino acid sequence having atleast 70%, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequenceidentity to one of SEQ ID NOs 5, 21, 22, 23; or 6, 21, 22, 23; or 7, 21,22, 24; or 8, 21, 22, 25; or 35, 21, 22, 25, or to one of the amino acidsequences of the V_(H) chain amino acid sequence shown in FIG. 2).

The antibody may optionally bind PD-L1, optionally human or murinePD-L1. The antibody may optionally have amino acid sequence componentsas described above. The antibody may be an IgG. In one embodiment an invitro complex, optionally isolated, comprising an antibody, or antigenbinding fragment, as described herein, bound to PD-L1 is provided.

The antibody may optionally inhibit or prevent interaction or functionalassociation between human PD-1 and human PD-L1, or between murine PD-1and murine PD-L1. Such inhibition or prevention of interaction orfunctional association between PD-1 and PD-L1 may inhibit or preventPD-L1-mediated activation of PD-1 or PD-L1/PD-1 signalling.

In one aspect of the present invention an isolated heavy chain variableregion polypeptide is provided, the heavy chain variable regionpolypeptide comprising the following CDRs:

(SEQ ID NO: 21) HC-CDR1: SYAIS (SEQ ID NO: 22)HC-CDR2: RIIPILGIANYAQKFQG (SEQ ID NO: 26)HC-CDR3: X₁X₂X₃X₄X₅X₆SX₇X₈AFDX₉where X₁=absent or G, X₂=G or S, X₃=G or Y, X₄═S, G or H, X₅═Y or G,X₅=G, N or Y, X₇=L or Y, X₈═Y or G, X₉═I or Y.

In some embodiments HC-CDR3 is one of GGSYGSLYAFDI (SEQ ID NO:23),GGYGGNSLYAFDI (SEQ ID NO:24) or SGHGYSYGAFDY (SEQ ID NO:25).

In one aspect of the present invention an antibody, or antigen bindingfragment, is provided, the antibody, or antigen binding fragment,comprising a heavy chain and a light chain variable region sequence,wherein:

-   -   the heavy chain comprises a HC-CDR1, HC-CDR2, HC-CDR3, having at        least 85% overall sequence identity to HC-CDR1: SYAIS (SEQ ID        NO:21), HC-CDR2 RIIPILGIANYAQKFQG (SEQ ID NO:22), HC-CDR3: one        of X₁X₂X₃X₄X₅X₆X₇X₈AFDX₉ (SEQ ID NO:26) or GGSYGSLYAFDI (SEQ ID        NO:23) or GGYGGNSLYAFDI (SEQ ID NO:24) or SGHGYSYGAFDY (SEQ ID        NO:25), respectively, where X₁=absent or G, X₂=G or S, X₃=G or        Y, X₄═S, G or H, X₅═Y or G, X₆=G, N or Y, X₇=L or Y, X₈═Y or G,        X₉═I or Y, and    -   the light chain comprises a LC-CDR1, LC-CDR2, LC-CDR3, having at        least 85% overall sequence identity to LC-CDR1: one of        SGRSSNIASHDVF (SEQ ID NO:9), GGDNIGRKSVH (SEQ ID NO:12),        SGSSSNIGNNYVS (SEQ ID NO:15), or TGSSSNIGAGYDVH (SEQ ID NO:18),        LC-CDR2: one of ETNKRPW (SEQ ID NO:10), DDGDRPS (SEQ ID NO:13),        DNNERLS (SEQ ID NO:16), or GNSNRPS (SEQ ID NO:19), LC-CDR3: one        of GAWDSGLTGML (SEQ ID NO:11), QAWDSTVV (SEQ ID NO:14),        GTWDSSLSVVV (SEQ ID NO:17), or QSYDSSLSGSYVV (SEQ ID NO:20).

In some embodiments the degree of sequence identity may be one of 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%.

In another aspect of the present invention an antibody, or antigenbinding fragment, optionally isolated, is provided comprising a heavychain and a light chain variable region sequence, wherein:

-   -   the heavy chain sequence has at least 85% sequence identity to        the heavy chain sequence of SEQ ID NO:5, 6, 7, 8 or 35 (FIG. 2),        and    -   the light chain sequence has at least 85% sequence identity to        the light chain sequence: SEQ ID NO:1, 2, 3 or 4 (FIG. 1). In        some embodiments the degree of sequence identity may be one of        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        99%, or 100%.

In some embodiments the antibody, antigen binding fragment, orpolypeptide further comprises variable region heavy chain frameworksequences between the CDRs according to the arrangementHCFR1:HC-CDR1:HCFR2:HC-CDR2:HCFR3:HC-CDR3:HCFR4. The framework sequencesmay be derived from human consensus framework sequences.

In one aspect of the present invention an isolated heavy chain variableregion polypeptide, optionally in combination with a light chainvariable region polypeptide as described herein, is provided, the heavychain variable region polypeptide comprising the following CDRs:

(SEQ ID NO: 21) HC-CDR1: SYAIS (SEQ ID NO: 22)HC-CDR2: RIIPILGIANYAQKFQG (SEQ ID NO: 26)HC-CDR3: X₁X₂X₃X₄X₅X₆SX₇X₈AFDX₉where X₁=absent or G, X₂=G or S, X₃=G or Y, X₄═S, G or H, X₅═Y or G,X₅=G, N or Y, X₇=L or Y, X₈═Y or G, X₉═I or Y.

In some embodiments HC-CDR3 is GGSYGSLYAFDI (SEQ ID NO:23) orGGYGGNSLYAFDI (SEQ ID NO:24) or SGHGYSYGAFDY (SEQ ID NO:25).

In some embodiments the antibody, antigen binding fragment, orpolypeptide further comprises variable region light chain frameworksequences between the CDRs according to the arrangementLCFR1:LC-CDR1:LCFR2:LC-CDR2:LCFR3:LC-CDR3:LCFR4. The framework sequencesmay be derived from human consensus framework sequences.

In some embodiments, the antibody, or antibody binding fragment, mayfurther comprise a human constant region. For example selected from oneof IgG1, IgG2, IgG3 and IgG4.

In some embodiments, the antibody, or antibody binding fragment, mayfurther comprise a murine constant region. For example, selected fromone of IgG1, IgG2A, IgG2B and IgG3.

In another aspect of the present invention, an antibody or antigenbinding fragment, optionally isolated, which is capable of binding toPD-L1, which is a bispecific antibody or a bispecific antigen bindingfragment is provided. The bispecific antibody or antigen bindingfragment comprises (i) an antigen binding fragment or polypeptidecapable of binding to PD-L1 as described herein, and (ii) an antigenbinding fragment which is capable of binding to a target protein otherthan PD-L1.

In some embodiments, the target protein other than PD-L1 may be a cellsurface receptor, e.g. a receptor expressed on the cell surface of Tcells. In some embodiments the cell surface receptor may be an immunecheckpoint receptor, e.g. a costimulatory receptor or an inhibitoryreceptor. In some embodiments, the costimulatory receptor may beselected from CD27, CD28, ICOS, CD40, CD122, OX40, 4-1BB and GITR. Insome embodiments, the inhibitory receptor may be selected from LAG-3,B7-H3, B7-H4, BTLA, CTLA-4, A2AR, VISTA, TIM-3, PD-1, and KIR.

In some embodiments, the target protein other than PD-L1 may be a cancermarker whose expression is associated with a cancer. In someembodiments, the cancer marker may be expressed at the cell surface. Insome embodiments, cancer marker may be selected from HER-2, HER-3, EGFR,EpCAM, CD30, CD33, CD38, CD20, CD24, CD90, CD15, CD52, CA-125, CD34,CA-15-3, CA-19-9, CEA, CD99, CD117, CD31, CD44, CD123, CD133, ABCB5 andCD45.

In another aspect of the present invention, a composition, e.g. apharmaceutical composition or medicament, is provided. The compositionmay comprise an antibody, antigen binding fragment, or polypeptide asdescribed herein and at least one pharmaceutically-acceptable carrier,excipient, adjuvant or diluent.

In another aspect of the present invention an isolated nucleic acidencoding an antibody, antigen binding fragment, or polypeptide asdescribed herein is provided. The nucleic acid may have a sequence ofone of SEQ ID NOs 27, 28, 29, 30, 31, 32, 33, 34 or 36 (FIG. 4), or acoding sequence which is degenerate as a result of the genetic code, ormay have a nucleotide sequence having at least 70% identity thereto,optionally one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%.

In one aspect of the present invention there is provided a vectorcomprising a nucleic acid described herein. In another aspect of thepresent invention, there is provided a host cell comprising the vector.For example, the host cell may be eukaryotic, or mammalian, e.g. ChineseHamster Ovary (CHO), or human or may be a prokaryotic cell, e.g. E.coli. In one aspect of the present invention a method for making anantibody, or antigen binding fragment or polypeptide as described hereinis provided, the method comprising culturing a host cell as describedherein under conditions suitable for the expression of a vector encodingthe antibody, or antigen binding fragment or polypeptide, and recoveringthe antibody, or antigen binding fragment or polypeptide.

In another aspect of the present invention an antibody, antigen bindingfragment or polypeptide is provided for use in therapy, or in a methodof medical treatment. In another aspect of the present invention anantibody, antigen binding fragment or polypeptide as described herein isprovided for use in the treatment of a T-cell dysfunctional disorder. Inanother aspect of the present invention, the use of an antibody, antigenbinding fragment or polypeptide as described herein in the manufactureof a medicament or pharmaceutical composition for use in the treatmentof a T-cell dysfunctional disorder is provided.

In another aspect of the present invention a method of enhancing T-cellfunction comprising administering an antibody, antigen binding fragmentor polypeptide as described herein to a dysfunctional T-cell isprovided. The method may be performed in vitro or in vivo.

In another aspect of the present invention a method of treating a T-celldysfunctional disorder is provided, the method comprising administeringan antibody, antigen binding fragment or polypeptide as described hereinto a patient suffering from a T-cell dysfunctional disorder.

In another aspect of the present invention an antibody, antigen bindingfragment or polypeptide is provided for use in the treatment of acancer. In another aspect of the present invention, the use of anantibody, antigen binding fragment or polypeptide as described herein inthe manufacture of a medicament or pharmaceutical composition for use inthe treatment of a cancer is provided.

In another aspect of the present invention a method of killing a tumourcell is provided, the method comprising administering an antibody,antigen binding fragment or polypeptide as described herein to a tumourcell. The method may be performed in vitro or in vivo. Killing of atumour cell may, for example, be as a result of antibody dependentcell-mediated cytotoxicity (ADCC), complement dependent cytotoxicity(CDC), or through the action of a drug conjugated to the antibody,antigen binding fragment or polypeptide.

In another aspect of the present invention a method of treating a canceris provided, the method comprising administering an antibody, antigenbinding fragment or polypeptide as described herein to a patientsuffering from a cancer.

The cancer may be a cancer which overexpresses PD-L1, or may comprisecells which overexpress PD-L1. The cancer may be a colorectal carcinomaor melanoma.

In another aspect of the present invention a method of modulating animmune response in a subject is provided, the method comprisingadministering to the subject an antibody, antigen binding fragment orpolypeptide as described herein such that the immune response in thesubject is modulated.

In another aspect of the present invention a method of inhibiting growthof tumor cells is provided, comprising administering an antibody,antigen binding fragment or polypeptide as described herein. The methodmay be in vitro or in vivo. In some embodiments a method of inhibitinggrowth of tumor cells in a subject is provided, the method comprisingadministering to the subject a therapeutically effective amount of anantibody, antigen binding fragment or polypeptide as described herein.

In another aspect of the present invention a method is provided, themethod comprising contacting a sample containing, or suspected tocontain, PD-L1 with an antibody or antigen binding fragment, asdescribed herein, and detecting the formation of a complex of antibody,or antigen binding fragment, and PD-L1.

In another aspect of the present invention a method of diagnosing adisease or condition in a subject is provided, the method comprisingcontacting, in vitro, a sample from the subject with an antibody, orantigen binding fragment, as described herein, and detecting theformation of a complex of antibody, or antigen binding fragment, andPD-L1.

In a further aspect of the present invention the use of an antibody, orantigen binding fragment, as described herein, for the detection ofPD-L1 in vitro is provided. In another aspect of the present inventionthe use of an antibody, or antigen binding fragment, as describedherein, as an in vitro diagnostic agent is provided.

In methods of the present invention the antibody, antigen bindingfragment or polypeptide may be provided as a composition as describedherein.

In some embodiments the antibody may be clone A1, C2, C4, H12 or H12_GLas described herein.

Description

Antibodies

Antibodies according to the present invention preferably bind to PD-L1(the antigen), preferably human or murine PD-L1, optionally with a K_(D)in the range 0.1 to 4 nM.

Antibodies according to the present invention may be provided inisolated form.

Antibodies according to the present invention may exhibit least one ofthe following properties:

-   -   a) binds to human, mouse or cynomolgus macaque PD-L1 with a        K_(D) of 1 μM or less, preferably one of ≦10 nM, ≦1 nM ≦500 pM,        ≦400 pM or ≦300 pM;    -   b) does not substantially bind to human PD-1, PD-L2, TIM-3,        LAG3, ICOS, CTLA-4, BTLA or CD28    -   c) inhibits or prevents interaction between human PD-1 and human        PD-L1 or inhibits or prevents interaction between murine PD-1        and murine PD-L1;    -   d) increases T-cell proliferation in an Mixed Lymphocyte        Reaction (MLR) assay (e.g. see Bromelow et al J. Immunol        Methods, 2001 January 1; 247(1-2):1-8);    -   e) increases interferon-gamma production in an MLR assay;    -   f) increases interferon-gamma production by tumour infiltrating        lymphocytes ex vivo;    -   g) increases interferon-gamma production by lymphocytes in        response to infection    -   h) inhibits tumour growth, optionally in vivo;    -   i) binds to PD-L1 (optionally human PD-L1) with greater affinity        than, or with similar affinity to, affinity of binding by        atezolizumab (MPDL3280A; RG7446);    -   j) binds to PD-L1(optionally human PD-L1) with greater avidity        than, or with similar avidity to, avidity of binding by        atezolizumab;    -   k) inhibits or prevents interaction between PD-L1 and PD-1        (optionally human PD-L1 and human PD-1) to a greater extent        than, or to a similar extent to, inhibition/prevention of        interaction by atezolizumab.

By “antibody” we include a fragment or derivative thereof, or asynthetic antibody or synthetic antibody fragment.

In view of today's techniques in relation to monoclonal antibodytechnology, antibodies can be prepared to most antigens. Theantigen-binding portion may be a part of an antibody (for example a Fabfragment) or a synthetic antibody fragment (for example a single chainFv fragment [ScFv]). Suitable monoclonal antibodies to selected antigensmay be prepared by known techniques, for example those disclosed in“Monoclonal Antibodies: A manual of techniques”, H Zola (CRC Press,1988) and in “Monoclonal Hybridoma Antibodies: Techniques andApplications”, J G R Hurrell (CRC Press, 1982). Chimaeric antibodies arediscussed by Neuberger et al (1988, 8th International BiotechnologySymposium Part 2, 792-799).

Monoclonal antibodies (mAbs) are useful in the methods of the inventionand are a homogenous population of antibodies specifically targeting asingle epitope on an antigen.

Polyclonal antibodies are useful in the methods of the invention.Monospecific polyclonal antibodies are preferred. Suitable polyclonalantibodies can be prepared using methods well known in the art.

Antigen binding fragments of antibodies, such as Fab and Fab₂ fragmentsmay also be used/provided as can genetically engineered antibodies andantibody fragments. The variable heavy (V_(H)) and variable light(V_(L)) domains of the antibody are involved in antigen recognition, afact first recognised by early protease digestion experiments. Furtherconfirmation was found by “humanisation” of rodent antibodies. Variabledomains of rodent origin may be fused to constant domains of humanorigin such that the resultant antibody retains the antigenicspecificity of the rodent parent antibody (Morrison et al (1984) Proc.Natl. Acad. Sd. USA 81, 6851-6855).

That antigenic specificity is conferred by variable domains and isindependent of the constant domains is known from experiments involvingthe bacterial expression of antibody fragments, all containing one ormore variable domains. These molecules include Fab-like molecules(Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al(1988) Science 240, 1038); single-chain Fv (ScFv) molecules where theV_(H) and V_(L) partner domains are linked via a flexible oligopeptide(Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl.Acad. Sd. USA 85, 5879) and single domain antibodies (dAbs) comprisingisolated V domains (Ward et al (1989) Nature 341, 544). A general reviewof the techniques involved in the synthesis of antibody fragments whichretain their specific binding sites is to be found in Winter & Milstein(1991) Nature 349, 293-299.

By “ScFv molecules” we mean molecules wherein the V_(H) and V_(L)partner domains are covalently linked, e.g. by a flexible oligopeptide.

Fab, Fv, ScFv and dAb antibody fragments can all be expressed in andsecreted from E. coli, thus allowing the facile production of largeamounts of the said fragments.

Whole antibodies, and F(ab′)₂ fragments are “bivalent”. By “bivalent” wemean that the said antibodies and F(ab′)₂ fragments have two antigencombining sites. In contrast, Fab, Fv, ScFv and dAb fragments aremonovalent, having only one antigen combining site. Synthetic antibodieswhich bind to PD-L1 may also be made using phage display technology asis well known in the art.

The present application also provides an antibody or antigen bindingfragment which is capable of binding to PD-L1, and which is a bispecificantibody or a bispecific antigen binding fragment. In some embodiments,the bispecific antibody or bispecific antigen binding fragment may beisolated.

In some embodiments, the bispecific antibodies and bispecific antigenbinding fragments comprise an antigen binding fragment or a polypeptideaccording to the present invention.

In some embodiments, the bispecific antibodies and bispecific antigenbinding fragments comprise an antigen binding fragment capable ofbinding to PD-L1, wherein the antigen binding fragment which is capableof binding to PD-L1 comprises or consists of an antigen binding fragmentor a polypeptide according to the present invention.

In some embodiments the bispecific antibodies and bispecific antigenbinding fragments comprise an antigen binding fragment capable ofbinding to PD-L1, and an antigen binding fragment capable of binding toanother target protein.

The antigen binding fragment capable of binding to another targetprotein may be capable of binding to another protein other than PD-L1.

In some embodiments, the target protein may be a cell surface receptor.In some embodiments, the target protein may be a cell surface receptorexpressed on the cell surface of an immune cell, e.g. T cell. In someembodiments the cell surface receptor may be an immune checkpointreceptor. In some embodiments, the immune checkpoint receptor may be acostimulatory receptor. In some embodiments, the costimulatory receptormay be selected from CD27, CD28, ICOS, CD40, CD122, OX40, 4-1BB andGITR. In some embodiments, the immune checkpoint receptor may be aninhibitory receptor. In some embodiments, the inhibitory receptor may beselected from LAG-3, B7-H3, B7-H4, BTLA, CTLA-4, A2AR, VISTA, TIM-3,PD-1, and KIR.

In some embodiments, the target protein may be a cancer marker. That is,the target protein may be a protein whose expression (e.g. upregulatedexpression) is associated with a cancer. In some embodiments, the cancermarker may be expressed at the cell surface. In some embodiments thecancer marker may be a receptor. In some embodiments, the cancer markermay be selected from HER-2, HER-3, EGFR, EpCAM, CD30, CD33, CD38, CD20,CD24, CD90, CD15, CD52, CA-125, CD34, CA-15-3, CA-19-9, CEA, CD99,CD117, CD31, CD44, CD123, CD133, ABCB5 and CD45.

In some embodiments, the antigen binding fragment for CD27 may comprisethe CDRs, light and heavy chain variable domains or other CD27 bindingfragment of e.g. anti-CD27 antibody clone 0323 (Millipore) or varlilumab(Celldex Therapeutics). In some embodiments, the antigen bindingfragment for CD28 may comprise the CDRs, light and heavy chain variabledomains or other CD28 binding fragment of e.g. anti-CD28 antibody cloneCD28.6 (eBioscience), clone CD28.2, clone JJ319 (Novus Biologicals),clone 204.12, clone B-23, clone 10F3 (Thermo Scientific PierceAntibodies), clone 37407 (R&D Systems), clone 204-12 (AbnovaCorporation), clone 15E8 (EMD Millipore), clone 204-12, clone YTH913.12(AbD Serotec), clone B-T3 (Acris Antibodies), clone 9H6E2 (SinoBiological), clone C28/77 (MyBioSource.com), clone KOLT-2 (ALPCO), clone152-2E10 (Santa Cruz Biotechnology), or clone XPH-56 (CreativeDiagnostics). In some embodiments, the antigen binding fragment for ICOSmay comprise the CDRs, light and heavy chain variable domains or otherICOS binding fragment of e.g. anti-ICOS antibody clone ISA-3(eBioscience), clone SP98 (Novus Biologicals), clone 1G1, clone 3G4(Abnova Corporation), clone 669222 (R&D Systems), clone TQ09 (CreativeDiagnostics), or clone C398.4A (BioLegend). In some embodiments, theantigen binding fragment for CD40 may comprise the CDRs, light and heavychain variable domains or other CD40 binding fragment of e.g. anti-CD40antibody clone 82111 (R&D Systems), or ASKP1240 (Okimura et al., AM JTransplant (2014) 14(6) 1290-1299). In some embodiments, the antigenbinding fragment for CD122 may comprise the CDRs, light and heavy chainvariable domains or other CD122 binding fragment of anti-CD122 antibodyclone mikβ2 (PharMingen). In some embodiments, the antigen bindingfragment for OX40 may comprise the CDRs, light and heavy chain variabledomains or other OX40 binding fragment of e.g. anti-OX40 antibodiesdisclosed in US 20130280275, U.S. Pat. No. 8,283,450 or WO2013038191,e.g. clone 12H3 or clone 20E5. In some embodiments, the antigen bindingfragment for 4-1BB may comprise the CDRs, light and heavy chain variabledomains or other 4-1BB binding fragment of e.g. anti-4-1BB antibodyPF-05082566 (Fisher et al., Cancer Immunol Immunother (2012) 61:1721-1733), or urelumab (BMS-665513; Bristol-Myers Squibb; Li and Liu,Clin Pharmacol (2013); 5: 47-53). In some embodiments, the antigenbinding fragment for GITR may comprise the CDRs, light and heavy chainvariable domains or other GITR binding fragment of e.g. anti-GITRantibody TRX-518 (Tolerx^(R); Schaer et al., (2010) 11(12): 1378-1386),or clone AIT 518D (LifeSpan Biosciences). In some embodiments, theantigen binding fragment for LAG3 may comprise the CDRs, light and heavychain variable domains or other LAG3 binding fragment of e.g. anti-LAG3antibody clone 17B4 (Enzo Life Sciences), clone 333210 (R&D Systems), orclone 14L676 (United States Biological). In some embodiments, theantigen binding fragment for B7-H3 may comprise the CDRs, light andheavy chain variable domains or other B7-H3 binding fragment of e.g.anti-B7-H3 antibody clones disclosed in US 20130078234, WO2014160627 orWO2011109400. In some embodiments, the antigen binding fragment forB7-H4 may comprise the CDRs, light and heavy chain variable domains orother B7-H4 binding fragment of e.g. anti-B7-H4 antibody clonesdisclosed in WO2013067492, WO2009073533 or EP2934575, for example clone2H9. In some embodiments, the antigen binding fragment for BTLA maycomprise the CDRs, light and heavy chain variable domains or other BTLAbinding fragment of e.g. anti-BTLA antibody clone 1B7, clone 2G8, clone4C5 (Abnova Corporation), clone 4B8 (antibodies-online), clone MIH26(Thermo Scientific Pierce Antibodies), clone UMAB61 (OriGeneTechnologies), clone 330104 (R&D Systems), clone 1B4 (LifeSpanBioSciences), clone 440205, clone 5E7 (Creative Diagnostics). In someembodiments, the antigen binding fragment for CTLA4 may comprise theCDRs, light and heavy chain variable domains or other CTLA4 bindingfragment of e.g. anti-CTLA4 antibody clone 2F1, clone 1F4 (AbnovaCorporation), clone 9H10 (EMD Millipore), clone BNU3 (GeneTex), clone1E2, clone AS32 (LifeSpan BioSciences) clone A3.4H2.H12 (AcrisAntibodies), clone 060 (Sino Biological), clone BU5G3 (CreativeDiagnostics), clone MIH8 (MBL International), clone A3.6B10.G1, or cloneL3D10 (BioLegend). In some embodiments, the antigen binding fragment forA2AR may comprise the CDRs, light and heavy chain variable domains orother A2AR binding fragment of e.g. anti-A2AR antibody clone 7F6(Millipore; Koshiba et al. Molecular Pharmacology (1999); 55: 614-624.In some embodiments, the antigen binding fragment for VISTA may comprisethe CDRs, light and heavy chain variable domains or other VISTA bindingfragment of e.g. anti-VISTA antibodies disclosed in WO2015097536 orUS20140105912, e.g. clone 13F3. In some embodiments, the antigen bindingfragment for TIM-3 may comprise the CDRs, light and heavy chain variabledomains or other TIM-3 binding fragment of e.g. anti-TIM-3 antibodyclone F38-2E2 (BioLegend), clone 2E2 (Merck Millipore; Pires da Silva etal., Cancer Immunol Res (2014) 2(5): 410-422), clone 6B6E2, clone 024(Sino Biological) clone 344801 (R&D Systems), clone E-18, clone H-191(Santa Cruz Biotechnology), or clone 13A224 (United States Biological).In some embodiments, the antigen binding fragment for PD-1 may comprisethe CDRs, light and heavy chain variable domains or other PD-1 bindingfragment of e.g. anti-PD-1 antibody clone J116, clone MIH4(eBioscience), clone 7A11B1 (Rockland Immunochemicals Inc.), clone192106 (R&D Systems), clone J110, clone J105 (MBL International), clone12A7D7, clone 7A11B1 (Abbiotec), clone #9X21 (MyBioSource.com), clone4H4D1 (Proteintech Group), clone D3W4U, clone D3045 (Cell SignalingTechnology), clone RMP1-30, clone RMP1-14 (Merck Millipore), cloneEH12.2H7 (BioLegend), clone 10B1227 (United States Biological), cloneUMAB198, clone UMAB197 (Origene Technologies), nivolumab (BMS-936558),lambrolizumab, or anti-PD-1 antibodies described in WO 2010/077634 or WO2006/121168. In some embodiments, the antigen binding fragment for KIRmay comprise the CDRs, light and heavy chain variable domains or otherKIR binding fragment of e.g. anti-KIR antibody clone 1-7F9 (Romagne etal., Blood (2009) 114(13): 2667-2677), lirilumab (BMS-986015; Sola etal., J Immunother Cancer (2013); 1:P40) or anti-KIR antibodies describedin US 2015/0344576 or WO 2014/066532. In some embodiments, the antigenbinding fragment for HER-2 may comprise the CDRs, light and heavy chainvariable domains or other HER-2 binding fragment of e.g. anti-HER-2antibody trastuzumab (Herceptin), or anti-HER-2 antibodies described inWO 2003/006509 or WO 2008/019290. In some embodiments, the antigenbinding fragment for HER-3 may comprise the CDRs, light and heavy chainvariable domains or other HER-3 binding fragment of e.g. anti-HER-3antibody clone MM-121 (Lyu et al., Int. J Clin Exp Pathol (2015) 8(6):6143-6156), MEHD7945A (Schaefer et al., Cancer Cell (2011) 20(4):472-486), AMG 888 (U3-1287; Aurisicchio et al., Oncotarget (2012) 3(8):744-758) or anti-HER-3 antibodies described in WO2008/100624 or WO2013048883. In some embodiments, the antigen binding fragment for EGFRmay comprise the CDRs, light and heavy chain variable domains or otherEGFR binding fragment of e.g. anti-EGFR antibody panitumumab (ABX-EGF;Vectibix), cetuximab (Erbitux), nimotuzumab, matazumab (EMD 7200) orantibody clone 048-006 (Sogawa et al., Nucl Med Comm (2012) 33(7):719-725). In some embodiments, the antigen binding fragment for EpCAMmay comprise the CDRs, light and heavy chain variable domains or otherEpCAM binding fragment of e.g. anti-EpCAM antibody edrecolomab, ING-1,3622W4, or adecatumumab (Munz et al., Cancer Cell Int (2010) 10:44). Insome embodiments, the antigen binding fragment for CD30 may comprise theCDRs, light and heavy chain variable domains or other CD30 bindingfragment of e.g. anti-CD30 antibody brentuximab (cAC10), clone SGN-30(Wahl et al., Cancer Res 2002 62(13):3736-3742), clone 5F11 (Borchmannet al., Blood (2003) 102(1): 3737-3742), or anti-CD30 antibodiesdescribed in WO 1993024135 or WO 2003059282. In some embodiments, theantigen binding fragment for CD33 may comprise the CDRs, light and heavychain variable domains or other CD33 binding fragment of e.g. anti-CD33antibody lintuzumab (SGN-33), gemtuzumab (Mylotarg), or clone hP67.7(Sievers et al., Blood (1999) 93(11): 3678-3684). In some embodiments,the antigen binding fragment for CD38 may comprise the CDRs, light andheavy chain variable domains or other CD38 binding fragment of e.g.anti-CD38 antibody daratumumab (Darzalex), SAR650984 (Martin et al., JClin Oncol (2014) 32:5s, (suppl; abstr 8532) or MOR202 (MorphoSys AG),or anti-CD38 antibodies described in WO 2006099875 or US 20100285004. Insome embodiments, the antigen binding fragment for CD20 may comprise theCDRs, light and heavy chain variable domains or other CD20 bindingfragment of e.g. anti-CD20 antibody rituximab, ocrelizumab, ofatumumab,obinutuzumab or BM-ca (Kobayashi et al., Cancer Med (2013) 2(2):130-143). In some embodiments, the antigen binding fragment for CD24 maycomprise the CDRs, light and heavy chain variable domains or other CD24binding fragment of e.g. anti-CD24 antibody clone eBioSN3 (eBioscience),clone ML5 (BD Biosciences), or anti-CD24 antibodies described in WO2008059491. In some embodiments, the antigen binding fragment for CD90may comprise the CDRs, light and heavy chain variable domains or otherCD90 binding fragment of e.g. anti-CD90 antibody clone 5E10 (BDBiosciences). In some embodiments, the antigen binding fragment for CD15may comprise the CDRs, light and heavy chain variable domains or otherCD15 binding fragment of e.g. anti-CD15 antibody clone C3D-1, Carb-3(DAKO A/S), MMA (Roche) or BY87 (Abcam). In some embodiments, theantigen binding fragment for CD52 may comprise the CDRs, light and heavychain variable domains or other CD52 binding fragment of e.g. anti-CD52antibody alemtuzumab, clone H1186, or clone YTH34.5 (AbD Serotec). Insome embodiments, the antigen binding fragment for CA-125 may comprisethe CDRs, light and heavy chain variable domains or other CA-125 bindingfragment of e.g. anti-CA-125 antibody oregovomab. In some embodiments,the antigen binding fragment for CD34 may comprise the CDRs, light andheavy chain variable domains or other CD34 binding fragment of e.g.anti-CD34 antibody clone 561 (BioLegend), clone 581 (Beckton Dickinson),or clone 5F3 (Sigma Aldrich). In some embodiments, the antigen bindingfragment for CA-15-3 may comprise the CDRs, light and heavy chainvariable domains or other CA-15-3 binding fragment of e.g. anti-CA-15-3antibody clone 2F16 (USBiological), clone TA998 (ThermoFisherScientific), clone 1D1 (Sigma Aldrich), or Mab AR20.5 (Qi et al., HybridHybridomics (2001) 20(5-6): 313-324). In some embodiments, the antigenbinding fragment for CA-19-9 may comprise the CDRs, light and heavychain variable domains or other CA-19-9 binding fragment of e.g.anti-CA-19-9 antibody clone 116-NS-19-9 (DAKO A/S), clone SPM110, orclone 121SLE (ThermoFisher Scientific). In some embodiments, the antigenbinding fragment for CEA may comprise the CDRs, light and heavy chainvariable domains or other CEA binding fragment of e.g. anti-CEA antibodylabetuzumab, C2-45 (Kyowa Hakko Kirin Co. Ltd.) or anti-CEA antibodiesdisclosed in Imakiire et al., Int J Cancer (2004) 108: 564-570 or WO2011034660. In some embodiments, the antigen binding fragment for CD99may comprise the CDRs, light and heavy chain variable domains or otherCD99 binding fragment of e.g. anti-CD99 antibody clone C7A (Moricoli etal., J Immunol Methods (2014) 408: 35-45) or clone 12E7 (DAKO A/S). Insome embodiments, the antigen binding fragment for CD117 may comprisethe CDRs, light and heavy chain variable domains or other CD117 bindingfragment of e.g. anti-CD117 antibody clone CK6 (Lebron et al., CancerBiot Ther (2014) 15(9): 1208-1218), or clone 104D2 (Sigma Aldrich). Insome embodiments, the antigen binding fragment for CD31 may comprise theCDRs, light and heavy chain variable domains or other CD31 bindingfragment of e.g. anti-CD31 antibody clone JC70A (DAKO A/S). In someembodiments, the antigen binding fragment for CD44 may comprise theCDRs, light and heavy chain variable domains or other CD44 bindingfragment of e.g. anti-CD44 antibody PF-03475952 (Runnels et al., AdvTher (2010); 27(3): 168-180), RG7356 (Vugts et al., MAbs (2014) 6(2):567-575), clone IM7, or clone A3D8 (Sigma Aldrich). In some embodiments,the antigen binding fragment for CD123 may comprise the CDRs, light andheavy chain variable domains or other CD123 binding fragment of e.g.anti-CD123 antibody CSL362 (Nievergall et al., Blood (2014)123(8):1218-1228), CSL360 (He et al., Leuk Lymphoma (2015) 56(5):1406-1415) 73G (Jin et al., Cell Stem Cell (2009) 5(1): 31-42) clone 6H6(AbD Serotec) or anti-CD123 antibodies described in WO 2014130635. Insome embodiments, the antigen binding fragment for CD133 may comprisethe CDRs, light and heavy chain variable domains or other CD133 bindingfragment of e.g. anti-CD133 antibody clone 6B3, clone 9G4, clone AC141(Wang et al., Hybridoma (Larchmt) (2010) 29(3): 241-249), clone 6B6(Chen et al., Hybridoma (Larchmt) (2010) 29(4): 305-310, clone AC113(Miltenyi Biotec), or anti-CD133 antibodies described in WO 2011149493.In some embodiments, the antigen binding fragment for ABCB5 may comprisethe CDRs, light and heavy chain variable domains or other ABCB5 bindingfragment of e.g. anti-ABCB5 antibody clone 5H3C6 (Thermo FisherScientific). In some embodiments, the antigen binding fragment for CD45may comprise the CDRs, light and heavy chain variable domains or otherCD45 binding fragment of e.g. anti-CD45 antibody YAML568 (Glatting etal., J Nucl Med (2006) 47(8): 1335-1341) or clone BRA-55 (SigmaAldrich).

An antigen binding fragment of a bispecific antibody or bispecificantigen binding fragment according to the present invention may be anyfragment of a polypeptide which is capable of binding to an antigen. Insome embodiments, an antigen binding fragment comprises at least thethree light chain CDRs (i.e. LC-CDR1, LC-CDR2 and LC-CDR3) and threeheavy chain CDRs (i.e. HC-CDR1, HC-CDR2 and HC-CDR3) which togetherdefine the antigen binding region of an antibody or antigen bindingfragment. In some embodiments, an antigen binding fragment may comprisethe light chain variable domain and heavy chain variable domain of anantibody or antigen binding fragment. In some embodiments, an antigenbinding fragment may comprise the light chain polypeptide and heavychain polypeptide of an antibody or antigen binding fragment.

Bispecific antibodies and bispecific antigen binding fragments accordingto the invention may be provided in any suitable format, such as thoseformats described in Kontermann MAbs 2012, 4(2): 182-197, which ishereby incorporated by reference in its entirety. For example, abispecific antibody or bispecific antigen binding fragment may be abispecific antibody conjugate (e.g. an IgG2, F(ab′)₂ or CovX-Body), abispecific IgG or IgG-like molecule (e.g. an IgG, scFv₄-Ig, IgG-scFv,scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, 2 in 1-IgG, mAb², or Tandemab commonLC), an asymmetric bispecific IgG or IgG-like molecule (e.g. a kih IgG,kih IgG common LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pair orSEED-body), a small bispecific antibody molecule (e.g. a Diabody (Db),dsDb, DART, scDb, tandAbs, tandem scFv (taFv), tandem dAb/VHH, triplebody, triple head, Fab-scFv, or F(ab′)₂-scFv₂), a bispecific Fc andC_(H)3 fusion protein (e.g. a taFv-Fc, Di-diabody, scDb-C_(H)3,scFv-Fc-scFv, HCAb-VHH, scFv-kih-Fc, or scFv-kih-C_(H)3), or abispecific fusion protein (e.g. a scFv₂-albumin, scDb-albumin,taFv-toxin, DNL-Fab₃, DNL-Fab₄-IgG, DNL-Fab₄-IgG-cytokine₂). See inparticular FIG. 2 of Kontermann MAbs 2012, 4(2): 182-19.

The skilled person is able to design and prepare bispecific antibodiesand bispecific antigen binding fragments according to the presentinvention.

Methods for producing bispecific antibodies include chemicallycrosslinking of antibodies or antibody fragments, e.g. with reducibledisulphide or non-reducible thioether bonds, for example as described inSegal and Bast, 2001. Production of Bispecific Antibodies. CurrentProtocols in Immunology. 14:IV:2.13:2.13.1-2.13.16, which is herebyincorporated by reference in its entirety. For example,N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used tochemically crosslink e.g. Fab fragments via hinge region SH-groups, tocreate disulfide-linked bispecific F(ab)₂ heterodimers.

Other methods for producing bispecific antibodies include fusingantibody-producing hybridomas e.g. with polyethylene glycol, to producea quadroma cell capable of secreting bispecific antibody, for example asdescribed in D. M. and Bast, B. J. 2001. Production of BispecificAntibodies. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16.

Bispecific antibodies and bispecific antigen binding fragments accordingto the present invention can also be produced recombinantly, byexpression from e.g. a nucleic acid construct encoding polypeptides forthe antigen binding molecules, for example as described in AntibodyEngineering: Methods and Protocols, Second Edition (Humana Press, 2012),at Chapter 40: Production of Bispecific Antibodies: Diabodies and TandemscFv (Hornig and Farber-Schwarz), or French, How to make bispecificantibodies, Methods Mol. Med. 2000; 40:333-339, the entire contents ofboth of which are hereby incorporated by reference. For example, a DNAconstruct encoding the light and heavy chain variable domains for thetwo antigen binding fragments (i.e. the light and heavy chain variabledomains for the antigen binding fragment capable of binding PD-L1, andthe light and heavy chain variable domains for the antigen bindingfragment capable of binding to another target protein), and includingsequences encoding a suitable linker or dimerization domain between theantigen binding fragments can be prepared by molecular cloningtechniques. Recombinant bispecific antibody can thereafter be producedby expression (e.g. in vitro) of the construct in a suitable host cell(e.g. a mammalian host cell), and expressed recombinant bispecificantibody can then optionally be purified.

Antibodies may be produced by a process of affinity maturation in whicha modified antibody is generated that has an improvement in the affinityof the antibody for antigen, compared to an unmodified parent antibody.Affinity-matured antibodies may be produced by procedures known in theart, e.g., Marks et al., Rio/Technology 10:779-783 (1992); Barbas et al.Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995);Jackson et al., J. Immunol. 154(7):331 0-15 9 (1995); and Hawkins et al,J. Mol. Biol. 226:889-896 (1992).

Antibodies according to the present invention preferably exhibitspecific binding to PD-L1. An antibody that specifically binds to atarget molecule preferably binds the target with greater affinity,and/or with greater duration than it binds to other targets. The presentantibodies may bind with greater affinity to PD-L1 than to anothermember of the CD28 family. In some embodiments the present antibodiesmay bind with greater affinity to PD-L1 than to one or more of PD-L2,TIM-3, LAG-3, ICOS, BTLA, CD28 or CTLA-4. In one embodiment, the extentof binding of an antibody to an unrelated target is less than about 10%of the binding of the antibody to the target as measured, e.g., byELISA, or by a radioimmunoassay (RIA). Alternatively, the bindingspecificity may be reflected in terms of binding affinity where theanti-PD-L1 antibody of the present invention binds to PD-L1 with a K_(D)that is at least 0.1 order of magnitude (i.e. 0.1×10^(n), where n is aninteger representing the order of magnitude) greater than the K_(D) ofthe antibody towards another target molecule, e.g. another member of theCD28 family. This may optionally be one of at least 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

Antibodies according to the present invention preferably have adissociation constant (K_(D)) of one of ≦10 nM, ≦1 nM ≦500 pM, ≦400 pMor ≦300 pM. The K_(D) may be in the range about 0.1 to about 4 nM.Binding affinity of an antibody for its target is often described interms of its dissociation constant (K_(D)). Binding affinity can bemeasured by methods known in the art, such as by Surface PlasmonResonance (SPR), or by a radiolabeled antigen binding assay (RIA)performed with the Fab version of the antibody and antigen molecule.

Antibodies according to the present invention preferably exhibit bindingto PD-L1 (e.g. human PD-L1) with greater affinity than, or with similaraffinity to, affinity of binding by atezolizumab (MPDL3280A; RG7446).

As used herein, an antibody displaying ‘greater affinity’ for a giventarget molecule compared to a reference antibody binds to that targetmolecule with greater strength as compared to the strength of binding ofthe reference antibody to the target molecule. The affinity of anantibody for a given target molecule can be determined quantitatively.

Relative affinity of binding of an antibody according the invention toPD-L1 compared to atezolizumab can be determined for example by ELISA,as described herein. In some embodiments, an antibody according to thepresent invention may have a dissociation constant (K_(D)) for PD-L1which is less than or equal to the K_(D) of atezolizumab for PD-L1.

In some embodiments, an antibody according the present invention mayhave affinity for PD-L1 which is 1.01 times or greater, 1.05 times orgreater, 1.1 times or greater, 1.15 times or greater, 1.2 times orgreater, 1.25 times or greater, 1.3 times or greater, 1.35 times orgreater, 1.4 times or greater, 1.45 times or greater, 1.5 times orgreater than the affinity of atezolizumab for PD-L1, in a given assay.In some embodiments, an antibody according the present invention maybind to PD-L1 with a K_(D) value which is 0.99 times or less, 0.95 timesor less, 0.9 times or less, 0.85 times or less, 0.8 times or less, 0.75times or less, 0.7 times or less, 0.65 times or less, 0.6 times or less,0.55 times or less, 0.5 times or less of the K_(D) value of atezolizumabfor PD-L1, in a given assay.

Antibodies according to the present invention preferably exhibit bindingto PD-L1 (e.g. human PD-L1) with greater avidity than, or with similaravidity to, avidity of binding by atezolizumab.

As used herein, an antibody displaying ‘greater avidity’ for a giventarget molecule compared to a reference antibody binds to that targetmolecule to form a stronger antibody:target complex (e.g. a more stableantibody:target complex) as compared to the antibody:target complexformed by binding of the reference antibody to the target molecule.Avidity of an antibody for a given target molecule can be determinedquantitatively.

Relative avidity of binding of an antibody according the invention toPD-L1 compared to atezolizumab can be determined for example by ELISA,as described herein. In some embodiments, an antibody according to thepresent invention may have an avidity of binding for PD-L1 which isgreater than or equal to the avidity of binding of atezolizumab forPD-L1.

In some embodiments, an antibody according the present invention mayhave avidity of binding for PD-L1 which is 1.01 times or greater, 1.05times or greater, 1.1 times or greater, 1.15 times or greater, 1.2 timesor greater, 1.25 times or greater, 1.3 times or greater, 1.35 times orgreater, 1.4 times or greater, 1.45 times or greater, 1.5 times orgreater than avidity of binding of atezolizumab for PD-L1, in a givenassay.

Antibodies according to the present invention preferably inhibit orprevent interaction between PD-L1 and PD-1 (e.g. human PD-L1 and humanPD-1) to a greater extent than, or to a similar extent to,inhibition/prevention of interaction between PD-L1 and PD-1 byatezolizumab. Relative inhibition/prevention of interaction betweenPD-L1 and PD-1 of an antibody according the invention for PD-L1 comparedto atezolizumab can be determined for example by ELISA, as describedherein. In some embodiments, an antibody according to the presentinvention may inhibit/prevent interaction between PD-L1 and PD-1 to anextent which is greater than or equal to inhibition/prevention ofinteraction between PD-L1 and PD-1 by atezolizumab. In some embodiments,an antibody according the present invention may inhibit/preventinteraction between PD-L1 and PD-1 to an extent which is 1.01 times orgreater, 1.05 times or greater, 1.1 times or greater, 1.15 times orgreater, 1.2 times or greater, 1.25 times or greater, 1.3 times orgreater, 1.35 times or greater, 1.4 times or greater, 1.45 times orgreater, 1.5 times or greater than inhibition/prevention of interactionbetween PD-L1 and PD-1 by atezolizumab, in a given assay.

In some embodiments, an antibody according the present invention mayinhibit/prevent interaction between PD-L1 and PD-1 with a value for halfmaximal inhibition of interaction (i.e. an IC₅₀ value for inhibition ofinteraction between PD-L1 and PD-1) which is lower than the IC₅₀ valuefor inhibition of interaction between PD-L1 and PD-1 by atezolizumab. Insome embodiments, an antibody according the present invention mayinhibit/prevent interaction between PD-L1 and PD-1 with an IC₅₀ valuewhich is 0.99 times or less, 0.95 times or less, 0.9 times or less, 0.85times or less, 0.8 times or less, 0.75 times or less, 0.7 times or less,0.65 times or less, 0.6 times or less, 0.55 times or less, 0.5 times orless of the IC₅₀ value for inhibition of interaction between PD-L1 andPD-1 by atezolizumab, in a given assay.

Antibodies according to the present invention may be “antagonist”antibodies that inhibit or reduce a biological activity of the antigento which it binds. Blocking of interaction between PD-1 and PD-L1assists in the restoration of T-cell function by inhibiting theimmune-inhibitory signalling pathway mediated by PD-1.

PD-L1 has also been shown to bind to B7-1 (CD80), an interaction thatalso suppresses T-cell proliferation and cytokine production.

In some aspects, the antibody is clone A1, or a variant of A1. A1comprises the following CDR sequences:

Light chain:  (SEQ ID NO: 9) LC-CDR1: SGRSSNIASHDVF (SEQ ID NO: 10)LC-CDR2: ETNKRPW (SEQ ID NO: 11) LC-CDR3: GAWDSGLTGML Heavy chain: (SEQ ID NO: 21) HC-CDR1: SYAIS (SEQ ID NO: 22)HC-CDR2: RIIPILGIANYAQKFQG (SEQ ID NO: 23) HC-CDR3: GGSYGSLYAFDI.

CDR sequences determined by Kabat definition.

In some aspects, the antibody is clone C2, or a variant of C2. C2comprises the following CDR sequences:

Light chain:  (SEQ ID NO: 12) LC-CDR1: GGDNIGRKSVH (SEQ ID NO: 13)LC-CDR2: DDGDRPS (SEQ ID NO: 14) LC-CDR3: QAWDSTVV Heavy chain: (SEQ ID NO: 21) HC-CDR1: SYAIS (SEQ ID NO: 22)HC-CDR2: RIIPILGIANYAQKFQG (SEQ ID NO: 23) HC-CDR3: GGSYGSLYAFDI.

CDR sequences determined by Kabat definition.

In some aspects, the antibody is clone C4, or a variant of C4. C4comprises the following CDR sequences:

Light chain:  (SEQ ID NO: 15) LC-CDR1: SGSSSNIGNNYVS (SEQ ID NO: 16)LC-CDR2: DNNERLS (SEQ ID NO: 17) LC-CDR3: GTWDSSLSVVV Heavy chain: (SEQ ID NO: 21) HC-CDR1: SYAIS (SEQ ID NO: 22)HC-CDR2: RIIPILGIANYAQKFQG (SEQ ID NO: 24) HC-CDR3: GGYGGNSLYAFDI.

CDR sequences determined by Kabat definition.

In some aspects, the antibody is clone H12, or a variant of H12. In someaspects, the antibody is clone H12_GL, or a variant of H12_GL. H12 andH12_GL each comprise the following CDR sequences:

Light chain:  (SEQ ID NO: 18) LC-CDR1: TGSSSNIGAGYDVH (SEQ ID NO: 19)LC-CDR2: GNSNRPS (SEQ ID NO: 20) LC-CDR3: QSYDSSLSGSYVV Heavy chain: (SEQ ID NO: 21) HC-CDR1: SYAIS (SEQ ID NO: 22)HC-CDR2: RIIPILGIANYAQKFQG (SEQ ID NO: 25) HC-CDR3: SGHGYSYGAFDY.

CDR sequences determined by Kabat definition.

Antibodies according to the present invention may comprise the CDRs ofA1, C2, C4, H12, or H12_GL or one of SEQ ID NOs 1 and 5; 2 and 6; 3 and7; 4 and 8; or 4 and 35. In an antibody according to the presentinvention one or two or three or four of the six CDR sequences may vary.A variant may have one or two amino acid substitutions in one or two ofthe six CDR sequences.

Amino acid sequences of the V_(H) and V_(L) chains of anti-PD-L1 clonesare shown in FIGS. 1 and 2. The encoding nucleotide sequences are shownin FIG. 4.

The light and heavy chain CDRs may also be particularly useful inconjunction with a number of different framework regions. Accordingly,light and/or heavy chains having LC-CDR1-3 or HC-CDR1-3 may possess analternative framework region. Suitable framework regions are well knownin the art and are described for example in M. Lefranc & G. Le:franc(2001) “The Immunoglobulin FactsBook”, Academic Press, incorporatedherein by reference.

In this specification, antibodies may have V_(H) and/or V_(L) chainscomprising an amino acid sequence that has a high percentage sequenceidentity to one or more of the V_(H) and/or V_(L) amino acid sequencesof SEQ ID NOs 1 and 5; 2 and 6; 3 and 7; 4 and 8; or 4 and 35, or to oneor the amino acid sequences shown in FIGS. 1 and 2.

For example, antibodies according to the present invention includeantibodies that bind PD-L1 and have a V_(H) or V_(L) chain thatcomprises an amino acid sequence having at least 70%, more preferablyone of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the V_(H) orV_(L) chain amino acid sequence of one of SEQ ID NOs 1 to 8 and 35, orto one or the amino acid sequences shown in FIGS. 1 and 2.

Antibodies according to the present invention may be detectably labelledor, at least, capable of detection. For example, the antibody may belabelled with a radioactive atom or a coloured molecule or a fluorescentmolecule or a molecule which can be readily detected in any other way.Suitable detectable molecules include fluorescent proteins, luciferase,enzyme substrates, and radiolabels. The binding moiety may be directlylabelled with a detectable label or it may be indirectly labelled. Forexample, the binding moiety may be an unlabelled antibody which can bedetected by another antibody which is itself labelled. Alternatively,the second antibody may have bound to it biotin and binding of labelledstreptavidin to the biotin is used to indirectly label the firstantibody.

Methods of Detection

Antibodies, or antigen binding fragments, described herein may be usedin methods that involve the binding of the antibody or antigen bindingfragment to PD-L1. Such methods may involve detection of the boundcomplex of antibody, or antigen binding fragment, and PD-L1. As such, inone embodiment a method is provided, the method comprising contacting asample containing, or suspected to contain, PD-L1 with an antibody orantigen binding fragment as described herein and detecting the formationof a complex of antibody, or antigen binding fragment, and PD-L1.

Suitable method formats are well known in the art, includingimmunoassays such as sandwich assays, e.g. ELISA. The method may involvelabelling the antibody, or antigen binding fragment, or PD-L1, or both,with a detectable label, e.g. fluorescent, luminescent or radio-label.PD-L1 expression may be measured by immunohistochemistry (IHC), forexample of a tissue sample obtained by biopsy.

Methods of this kind may provide the basis of a method of diagnosis of adisease or condition requiring detection and or quantitation of PD-L1 orPD-1. Such methods may be performed in vitro on a patient sample, orfollowing processing of a patient sample. Once the sample is collected,the patient is not required to be present for the in vitro method ofdiagnosis to be performed and therefore the method may be one which isnot practised on the human or animal body.

Such methods may involve determining the amount of PD-L1 present in apatient sample. The method may further comprise comparing the determinedamount against a standard or reference value as part of the process ofreaching a diagnosis. Other diagnostic tests may be used in conjunctionwith those described here to enhance the accuracy of the diagnosis orprognosis or to confirm a result obtained by using the tests describedhere.

Cancer cells may exploit the PD-1 pathway to create an immunosuppressiveenvironment, by upregulating expression of PD-L1 and/or PD-1, allowingactivation of the inhibitory PD-1 receptor on any T cells thatinfiltrate the tumor microenvironment and thereby suppressing theiractivity. Upregulation of PD-L1 and/or PD-1 expression has beendemonstrated in many different cancer types, and high PD-L1/PD-1expression has also been linked to poor clinical outcomes.

The level of PD-L1 or PD-1 present in a patient sample may be indicativethat a patient may respond to treatment with an anti-PD-L1 antibody. Thepresence of a high level of PD-L1 or PD-1 in a sample may be used toselect a patient for treatment with an anti-PD-L1 antibody. Theantibodies of the present invention may therefore be used to select apatient for treatment with anti-PD-L1 therapy.

Detection in a sample of PD-L1 or PD-1 may be used for the purpose ofdiagnosis of a T-cell dysfunctional disorder or a cancerous condition inthe patient, diagnosis of a predisposition to a cancerous condition orfor providing a prognosis (prognosticating) of a cancerous condition.The diagnosis or prognosis may relate to an existing (previouslydiagnosed) cancerous condition, which may be benign or malignant, mayrelate to a suspected cancerous condition or may relate to the screeningfor cancerous conditions in the patient (which may be previouslyundiagnosed).

In one embodiment the level of PD-1 expression on CD8+ T cells may bedetected in order to indicate the degree of T-cell exhaustion andseverity of the disease state.

In one embodiment the level of PD-L1 expression, e.g. on antigenpresenting cells or tumor cells, may be detected in order to indicateexistence or severity of a disease state, for example of tissueinflammation or a cancer.

A sample may be taken from any tissue or bodily fluid. The sample maycomprise or may be derived from: a quantity of blood; a quantity ofserum derived from the individual's blood which may comprise the fluidportion of the blood obtained after removal of the fibrin clot and bloodcells; a tissue sample or biopsy; or cells isolated from saidindividual.

Methods according to the present invention are preferably performed invitro. The term “in vitro” is intended to encompass experiments withcells in culture whereas the term “in vivo” is intended to encompassexperiments with intact multi-cellular organisms.

Therapeutic Applications

Antibodies, antigen binding fragments and polypeptides according to thepresent invention and compositions comprising such agents may beprovided for use in methods of medical treatment. Treatment may beprovided to subjects having a disease or condition in need of treatment.The disease or condition may be one of a T-cell dysfunctional disorder,including a T-cell dysfunctional disorder associated with a cancer, or acancer, or a T-cell dysfunctional disorder associated with an infection,or an infection.

A T-cell dysfunctional disorder may be a disease or condition in whichnormal T-cell function is impaired causing downregulation of thesubject's immune response to pathogenic antigens, e.g. generated byinfection by exogenous agents such as microorganisms, bacteria andviruses, or generated by the host in some disease states such as in someforms of cancer (e.g. in the form of tumor associated antigens).

The T-cell dysfunctional disorder may comprise T-cell exhaustion orT-cell anergy. T-cell exhaustion comprises a state in which CD8+T-cellsfail to proliferate or exert T-cell effector functions such ascytotoxicity and cytokine (e.g. IFNγ) secretion in response to antigenstimulation. Exhausted T-cells may also be characterised by sustainedexpression of PD-1, where blockade of PD-1:PD-L1 interactions mayreverse the T-cell exhaustion and restore antigen-specific T cellresponses.

The T-cell dysfunctional disorder may be manifest as an infection, orinability to mount an effective immune response against an infection.The infection may be chronic, persistent, latent or slow, and may be theresult of bacterial, viral, fungal or parasitic infection. As such,treatment may be provided to patients having a bacterial, viral orfungal infection. Examples of bacterial infections include infectionwith Helicobacter pylori. Examples of viral infections include infectionwith HIV, hepatitis B or hepatitis C.

The T-cell dysfunctional disorder may be associated with a cancer, suchas tumor immune escape. Many human tumors express tumor-associatedantigens recognised by T cells and capable of inducing an immuneresponse. However, immune evasion is common and is believed to bemediated by a number of soluble factors, including PD-L1. As such,blocking the interaction of PD-1 and PD-L1 may inhibit this negativeimmunoregulatory signal to tumor cells and enhance tumor-specific CD8⁺T-cell immunity.

Cancers may also be treated where there is no indication of a T-celldysfunctional disorder such as T-cell exhaustion but the use of anantibody, antigen binding fragment or polypeptide according to thepresent invention allows the subject to suppress PD-1 signalling andmount an effective immune response with limited impairment, evasion orinduction of tumor immune escape. In such treatments, the antibody,antigen binding fragment or polypeptide may provide a treatment forcancer that involves prevention of the development of tumor immuneescape.

Cancers may also be treated which overexpress PD-L1. For example, suchtumor cells overexpressing PD-L1 may be killed directly by treatmentwith anti-PD-L1 antibodies, by antibody dependent cell-mediatedcytotoxicity (ADCC), complement dependent cytotoxicity (CDC), or usinganti-PD-L1 antibody-drug conjugates.

The treatment may be aimed at prevention of the T-cell dysfunctionaldisorder, e.g. prevention of infection or of the development orprogression of a cancer. As such, the antibodies, antigen bindingfragments and polypeptides may be used to formulate pharmaceuticalcompositions or medicaments and subjects may be prophylactically treatedagainst development of a disease state. This may take place before theonset of symptoms of the disease state, and/or may be given to subjectsconsidered to be at greater risk of infection or development of cancer.

Treatment may comprise co-therapy with a vaccine, e.g. T-cell vaccine,which may involve simultaneous, separate or sequential therapy, orcombined administration of vaccine and antibody, antigen bindingfragment or polypeptide in a single composition. In this context, theantibody, antigen binding fragment or polypeptide may be provided as anadjuvant to the vaccine. Limited proliferative potential of exhausted Tcells has been attributed as a main reason for failure of T-cellimmunotherapy and combination an agent capable of blocking or reversingT cell exhaustion is a potential strategy for improving the efficacy ofT-cell immunotherapy (Barber et al., Nature Vol 439, No. 9 p682-687February 2006).

Administration of an antibody, antigen binding fragment or polypeptideis preferably in a “therapeutically effective amount”, this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of the disease being treated. Prescription oftreatment, e.g. decisions on dosage etc., is within the responsibilityof general practitioners and other medical doctors, and typically takesaccount of the disorder to be treated, the condition of the individualpatient, the site of delivery, the method of administration and otherfactors known to practitioners. Examples of the techniques and protocolsmentioned above can be found in Remington's Pharmaceutical Sciences,20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

Formulating Pharmaceutically Useful Compositions and Medicaments

Antibodies, antigen binding fragments and polypeptides according to thepresent invention may be formulated as pharmaceutical compositions forclinical use and may comprise a pharmaceutically acceptable carrier,diluent, excipient or adjuvant.

In accordance with the present invention methods are also provided forthe production of pharmaceutically useful compositions, such methods ofproduction may comprise one or more steps selected from: isolating anantibody, antigen binding fragment or polypeptide as described herein;and/or mixing an isolated antibody, antigen binding fragment orpolypeptide as described herein with a pharmaceutically acceptablecarrier, adjuvant, excipient or diluent.

For example, a further aspect of the present invention relates to amethod of formulating or producing a medicament or pharmaceuticalcomposition for use in the treatment of a T-cell dysfunctional disorder,the method comprising formulating a pharmaceutical composition ormedicament by mixing an antibody, antigen binding fragment orpolypeptide as described herein with a pharmaceutically acceptablecarrier, adjuvant, excipient or diluent.

Infection

An infection may be any infection or infectious disease, e.g. bacterial,viral, fungal, or parasitic infection. In some embodiments it may beparticularly desirable to treat chronic/persistent infections, e.g.where such infections are associated with T cell dysfunction or T cellexhaustion.

It is well established that T cell exhaustion is a state of T celldysfunction that arises during many chronic infections (including viral,bacterial and parasitic), as well as in cancer (Wherry Nature ImmunologyVol.12, No.6, p492-499, June 2011).

An infection or infectious disease may be one in which PD-1 isupregulated (e.g. as reported by Radziewicz H, et al., J Virol. 2007;81(6):2545-2553 and Golden-Mason L et al., J Virol. 2007;81(17):9249-9258), thereby also implicating the PD-1:PD-L1 interactionas part of the disease state.

Examples of bacterial infections that may be treated include infectionby Bacillus spp., Bordetella pertussis, Clostridium spp.,Corynebacterium spp., Vibrio chloerae, Staphylococcus spp.,Streptococcus spp. Escherichia, Klebsiella, Proteus, Yersinia, Erwina,Salmonella, Listeria sp, Helicobacter pylori, mycobacteria (e.g.Mycobacterium tuberculosis) and Pseudomonas aeruginosa. For example, thebacterial infection may be sepsis or tuberculosis.

Yao et al. (PD-1 on dendritic cells impedes innate immunity againstbacterial infection. Blood 113(23):5811-5818 Jun. 4 2009) establishedPD-1 in the negative regulation of DC function during innate immuneresponse to infection by Listeria monocytogenes. Brahmamdam et al(Delayed administration of anti-PD-1 antibody reverses immunedysfunction and improves survival during sepsis. Journal of LeukocyteBiology vo.88, no.2 233-240, August 2010) reported that anti-PD-1antibody administered 24 h after sepsis prevented sepsis-induceddepletion of lymphocytes and DCs, increased Bcl-xL, blocked apoptosisand improved survival. Tim3:Galectin-9 interactions have been reportedto mediate T cell exhaustion and mediate the innate and adaptive immuneresponse to infection by Mycobacterium tuberculosis (Jayaraman et al.,The Journal of Immunology 2012, 188, 70.6).

Examples of viral infections that may be treated include infection byinfluenza virus, measles virus, hepatitis B virus (HBV), hepatitis Cvirus (HCV), human immunodeficiency virus (HIV), lymphocyticchoriomeningitis virus (LCMV), Herpes simplex virus and human papillomavirus.

Chronic viral infections, such as those caused by HCV, HBV, and HIVcommonly involve mechanisms to evade immune clearance. Expression ofPD-1 and TIM-3 have been identified as correlating with defective T cellresponses to hepatitis C virus (HCV) (McMahan et al., The Journal ofClinical Investigation Vol. 120, No. 12 p4546-4557, December 2010). InHCV, McMahan et al (supra) found that the level of dual TIM-3 and PD-1expression on HCV-specific CTLs predated the development of viralpersistence, providing prognostic information. Barber et al. (Nature Vol439, No. 9 p682-687 February 2006) reported that PD-1 is upregulatedduring chronic viral infection. In mice infected with LCMV they reportedthat blockade of the PD-1/PD-L1 inhibitory pathway had a beneficialeffect on CD8 T cells, restoring their ability to undergo proliferation,secrete cytokines, kill infected cells and decrease viral load. PD-1 isalso upregulated in HIV infection (Said et al., Nature Medicine Vol. 16,No.4 p452-460 April 2010). Blocking interaction between PD-1 and PD-L1contributed to viral clearance and improved T cell function in animalmodels of chronic viral infection (Said et al., supra).

Examples of fungal infections that may be treated include infection byAlternaria sp, Aspergillus sp, Candida sp and Histoplasma sp. The fungalinfection may be fungal sepsis or histoplasmosis.

Chang et al (Blockade of the negative co-stimulatory molecules PD-1 andCTLA-4 improves survival in primary and secondary fungal sepsis.Critical Care 2013, 17:R85) reported that anti-PD1 antibodies werehighly effective at improving survival in primary and secondary fungalsepsis. Lázár-Molnár et al (The PD-1/PD-L costimulatory pathwaycritically affects host resistance to the pathogenic fungus Histoplasmacapsulatum PNAS vol. 105, no.7, p2658-2663, 19 Feb. 2008) reported thatanti-PD-1 antibody significantly increased survival of mice infectedwith Histoplasma capsulatum. As such, the importance of T cellexhaustion in mediating fungal infection is well established.

Examples of parasitic infections that may be treated include infectionby Plasmodium species (e.g. Plasmodium falciparum, Plasmodium yoeli,Plasmodium ovale, Plasmodium vivax, or Plasmodium chabaudi chabaudi).The parasitic infection may be a disease such as malaria, leishmaniasisand toxoplasmosis.

Infection of humans with Plasmodium falciparum has been shown to resultin higher expression of PD-1 and T cell exhaustion mice (Butler et al.,Nature Immunology Vol.13, No.12, p 188-195 February 2012). Blockade ofPD-L1 and LAG-3 using anti-PD-L1 and anti-LAG-3 monoclonal antibodies invivo contributed to the restoration of CD4⁺ T-cell function,amplification of the number of follicular helper T cells,germinal-center B cells and plasmablasts, enhanced protective antibodiesand rapidly cleared blood-stage malaria in mice. It was also shown toblock the development of chronic infection (Butler et al.,supra).

Cancer

A cancer may be any unwanted cell proliferation (or any diseasemanifesting itself by unwanted cell proliferation), neoplasm or tumor orincreased risk of or predisposition to the unwanted cell proliferation,neoplasm or tumor. The cancer may be benign or malignant and may beprimary or secondary (metastatic). A neoplasm or tumor may be anyabnormal growth or proliferation of cells and may be located in anytissue. Examples of tissues include the adrenal gland, adrenal medulla,anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum,central nervous system (including or excluding the brain) cerebellum,cervix, colon, duodenum, endometrium, epithelial cells (e.g. renalepithelia), gallbladder, oesophagus, glial cells, heart, ileum, jejunum,kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node,lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx,omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervoussystem, peritoneum, pleura, prostate, salivary gland, sigmoid colon,skin, small intestine, soft tissues, spleen, stomach, testis, thymus,thyroid gland, tongue, tonsil, trachea, uterus, vulva, white bloodcells.

Tumors to be treated may be nervous or non-nervous system tumors.Nervous system tumors may originate either in the central or peripheralnervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma,ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma andoligodendroglioma. Non-nervous system cancers/tumors may originate inany other non-nervous tissue, examples include melanoma, mesothelioma,lymphoma, myeloma, leukemia, Non-Hodgkin's lymphoma (NHL), Hodgkin'slymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia(AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL),chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma,prostate carcinoma, breast cancer, lung cancer, colon cancer, ovariancancer, pancreatic cancer, thymic carcinoma, NSCLC, haematologic cancerand sarcoma.

Adoptive T Cell Transfer Therapy

Adoptive T cell transfer therapy generally refers to a process in whichwhite blood cells are removed from a subject, typically by drawing ablood sample from which white blood cells are separated, expanded invitro or ex vivo and returned either to the same subject or to adifferent subject. The treatment is typically aimed at increasing theamount/concentration of an active form of the required T cell populationin the subject. Such treatment may be beneficial in subjectsexperiencing T cell exhaustion.

Antibodies capable of blocking the mechanism of T cell exhaustion, orreversing it, provide a means of enhancing T cell activity and promotingT cell expansion.

Accordingly, in a further aspect of the present invention a method isprovided for expanding a population of T cells, wherein T cells arecontacted in vitro or ex vivo with an antibody, antigen binding fragmentor polypeptide according to the present invention.

The method may optionally comprise one or more of the following steps:taking a blood sample from a subject; isolating T cells from the bloodsample; culturing the T cells in in vitro or ex vivo cell culture (wherethey may be contacted with the antibody, antigen binding fragment orpolypeptide), collecting an expanded population of T cells; mixing the Tcells with an adjuvant, diluent, or carrier; administering the expandedT cells to a subject.

Accordingly, in some aspects of the present invention a method oftreatment of a subject having a T-cell dysfunctional disorder isprovided, the method comprising obtaining a blood sample from a subjectin need of treatment, culturing T cells obtained from the blood samplein the presence of an antibody, antigen binding fragment or polypeptideaccording to the present invention so as to expand the T cellpopulation, collecting expanded T cells, and administering the expandedT cells to a subject in need of treatment.

The T cells may be obtained from a subject requiring treatment, and maybe isolated and/or purified. They may be a CD4⁺ and/or CD8⁺ T-cellpopulation. The T-cells may represent a population experiencing T cellexhaustion and may optionally have upregulated expression of PD-1 and/orPD-L1.

During culture, T cells may be contacted with the antibody, antigenbinding fragment or polypeptide under conditions and for a period oftime suitable to allow expansion of the T cells to a desired number ofcells. After a suitable period of time the T cells may be harvested,optionally concentrated, and may be mixed with a suitable carrier,adjuvant or diluent and returned to the subject's body. A subject mayundergo one or more rounds of such therapy.

Methods of T cell expansion are well known in the art, such as thosedescribed in Kalamasz et al., J Immunother 2004 September-October;27(5):405-18; Montes et al., Clin Exp Immunol 2005 November;142(2):292-302; Wölfl and Greenburg Nature Protocols 9 p950-966 27 Mar.2014; Trickett and Kwan Journal of Immunological Methods Vol. 275,Issues 1-2, 1 Apr. 2003, p251-255; Butler et al PLoSONE 7(1) 12 Jan.2012.

Simultaneous or Sequential Administration

Compositions may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

In this specification an antibody, antigen binding fragment orpolypeptide of the present invention and an anti-infective agent orchemotherapeutic agent (therapeutic agent) may be administeredsimultaneously or sequentially.

In some embodiments, treatment with an antibody, antigen bindingfragment or polypeptide of the present invention may be accompanied bychemotherapy.

Simultaneous administration refers to administration of the antibody,antigen binding fragment or polypeptide and therapeutic agent together,for example as a pharmaceutical composition containing both agents(combined preparation), or immediately after each other and optionallyvia the same route of administration, e.g. to the same artery, vein orother blood vessel.

Sequential administration refers to administration of one of theantibody, antigen binding fragment or polypeptide or therapeutic agentfollowed after a given time interval by separate administration of theother agent. It is not required that the two agents are administered bythe same route, although this is the case in some embodiments. The timeinterval may be any time interval.

Anti-Infective Agents

In treating infection, an antibody, antigen binding fragment orpolypeptide of the present invention may be administered in combinationwith an anti-infective agent, as described above. The anti-infectiveagent may be an agent known to have action against the microorganism orvirus responsible for the infection.

Suitable anti-infective agents include antibiotics (such as penicillins,cephalosporins, rifamycins, lipiarmycins, quinolones, sulfonamides,macrolides, lincosamides, tetracyclines, cyclic lipopeptides,glycylcyclines, oxazolidinones, and lipiarmycins), anti-viral agents(such as reverse transcriptase inhibitors, integrase inhibitors,transcription factor inhibitors, antisense and siRNA agents and proteaseinhibitors), anti-fungal agents (such as polyenes, imidiazoles,triazoles, thiazoles, allylamines, and echinocandins) and anti-parasiticagents (such as antinematode agents, anticestode agents, antitrematodeagents, antiamoebic agents and antiprotozoal agents).

Chemotherapy

Chemotherapy refers to treatment of a cancer with a drug or withionising radiation (e.g. radiotherapy using X-rays or γ-rays). Inpreferred embodiments chemotherapy refers to treatment with a drug. Thedrug may be a chemical entity, e.g. small molecule pharmaceutical,antibiotic, DNA intercalator, protein inhibitor (e.g. kinase inhibitor),or a biological agent, e.g. antibody, antibody fragment, nucleic acid orpeptide aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide, orprotein. The drug may be formulated as a pharmaceutical composition ormedicament. The formulation may comprise one or more drugs (e.g. one ormore active agents) together with one or more pharmaceuticallyacceptable diluents, excipients or carriers.

A treatment may involve administration of more than one drug. A drug maybe administered alone or in combination with other treatments, eithersimultaneously or sequentially dependent upon the condition to betreated. For example, the chemotherapy may be a co-therapy involvingadministration of two drugs, one or more of which may be intended totreat the cancer.

The chemotherapy may be administered by one or more routes ofadministration, e.g. parenteral, intravenous injection, oral,subcutaneous, intradermal or intratumoral.

The chemotherapy may be administered according to a treatment regime.The treatment regime may be a pre-determined timetable, plan, scheme orschedule of chemotherapy administration which may be prepared by aphysician or medical practitioner and may be tailored to suit thepatient requiring treatment.

The treatment regime may indicate one or more of: the type ofchemotherapy to administer to the patient; the dose of each drug orradiation; the time interval between administrations; the length of eachtreatment; the number and nature of any treatment holidays, if any etc.For a co-therapy a single treatment regime may be provided whichindicates how each drug is to be administered.

Chemotherapeutic drugs and biologics may be selected from:

-   -   alkylating agents such as cisplatin, carboplatin,        mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide;    -   purine or pyrimidine anti-metabolites such as azathiopurine or        mercaptopurine;    -   alkaloids and terpenoids, such as vinca alkaloids (e.g.        vincristine, vinblastine, vinorelbine, vindesine),        podophyllotoxin, etoposide, teniposide, taxanes such as        paclitaxel (Taxol™), docetaxel;    -   topoisomerase inhibitors such as the type I topoisomerase        inhibitors camptothecins irinotecan and topotecan, or the type        II topoisomerase inhibitors amsacrine, etoposide, etoposide        phosphate, teniposide;    -   antitumor antibiotics (e.g. anthracyline antibiotics) such as        dactinomycin, doxorubicin (Adriamycin™), epirubicin, bleomycin,        rapamycin;    -   antibody based agents, such as anti-PD-1 antibodies, anti-TIM-3        antibodies, anti-CTLA-4, anti-LAG-3, anti-4-1BB, anti-GITR,        anti-CD27, anti-BLTA, anti-OX40, anti-VEGF, anti-TNFα,        anti-IL-2, antiGpIIb/IIIa, anti-CD-52, anti-CD20, anti-RSV,        anti-HER2/neu(erbB2), anti-TNF receptor, anti-EGFR antibodies,        monoclonal antibodies or antibody fragments, examples include:        cetuximab, panitumumab, infliximab, basiliximab, bevacizumab        (Avastin®), abciximab, daclizumab, gemtuzumab, alemtuzumab,        rituximab (Mabthera®), palivizumab, trastuzumab, etanercept,        adalimumab, nimotuzumab    -   EGFR inihibitors such as erlotinib, cetuximab and gefitinib    -   anti-angiogenic agents such as bevacizumab (Avastin®)    -   cancer vaccines such as Sipuleucel-T (Provenge®)

In one embodiment the chemotherapeutic agent is an anti-PD-1 antibody,anti-TIM-3 antibody, anti-CTLA-4, anti-LAG3, anti-41BB, anti-GITR,anti-CD27, anti-BLTA, anti-OX40, anti-VEGF, anti-TNFα, anti-IL2,anti-GpIIb/IIIa, anti-CD-52, anti-CD20, anti-RSV, anti-HER2/neu(erbB2),anti-TNF receptor, anti-EGFR or other antibody. In some embodiments, thechemotherapeutic agent is an immune checkpoint inhibitor orcostimulation molecule.

Further chemotherapeutic drugs may be selected from: 13-cis-RetinoicAcid, 2-Chlorodeoxyadenosine, 5-Azacitidine 5-Fluorouracil,6-Mercaptopurine, 6-Thioguanine, Abraxane, Accutane®, Actinomycin-DAdriamycin®, Adrucil®, Afinitor®, Agrylin®, Ala-Cort®, Aldesleukin,Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ®, Alkeran®,All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin,Amifostine, Aminoglutethimide, Anagrelide, Anandron®, Anastrozole,Arabinosylcytosine, Aranesp®, Aredia®, Arimidex®, Aromasin®, Arranon®,Arsenic Trioxide, Asparaginase, ATRA Avastin®, Azacitidine, BCG, BCNU,Bendamustine, Bevacizumab, Bexarotene, BEXXAR®, Bicalutamide, BiCNU,Blenoxane®, Bleomycin, Bortezomib, Busulfan, Busulfex®, CalciumLeucovorin, Campath®, Camptosar®, Camptothecin-11, Capecitabine, Carac™,Carboplatin, Carmustine, Casodex®, CC-5013, CCI-779, CCNU, CDDP, CeeNU,Cerubidine®, Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor,Cladribine, Cortisone, Cosmegen®, CPT-11, Cyclophosphamide, Cytadren®,Cytarabine Cytosar-U®, Cytoxan®, Dacogen, Dactinomycin, DarbepoetinAlfa, Dasatinib, Daunomycin, Daunorubicin, Daunorubicin Hydrochloride,Daunorubicin Liposomal, DaunoXome®, Decadron, Decitabine, Delta-Cortef®,Deltasone®, Denileukin, Diftitox, DepoCyt™, Dexamethasone, DexamethasoneAcetate, Dexamethasone Sodium Phosphate, Dexasone, Dexrazoxane, DHAD,DIC, Diodex, Docetaxel, Doxil®, Doxorubicin, Doxorubicin Liposomal,Droxia™, DTIC, DTIC-Dome®, Duralone®, Eligard™, Ellence™, Eloxatin™,Elspar®, Emcyt®, Epirubicin, Epoetin Alfa, Erbitux, Erlotinib, ErwiniaL-asparaginase, Estramustine, Ethyol Etopophos®, Etoposide, EtoposidePhosphate, Eulexin®, Everolimus, Evista®, Exemestane, Faslodex®,Femara®, Filgrastim, Floxuridine, Fludara®, Fludarabine, Fluoroplex®,Fluorouracil, Fluoxymesterone, Flutamide, Folinic Acid, FUDR®,Fulvestrant, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gleevec™,Gliadel® Wafer, Goserelin, Granulocyte-Colony Stimulating Factor,Granulocyte Macrophage Colony Stimulating Factor, Herceptin ®, Hexadrol,Hexalen®, Hexamethylmelamine, HMM, Hycamtin®, Hydrea®, HydrocortAcetate®, Hydrocortisone, Hydrocortisone Sodium Phosphate,Hydrocortisone Sodium Succinate, Hydrocortone Phosphate, Hydroxyurea,Ibritumomab, Ibritumomab Tiuxetan, Idamycin®, Idarubicin, Ifex®,IFN-alpha, Ifosfamide, IL-11, IL-2, Imatinib mesylate, ImidazoleCarboxamide, Interferon alfa, Interferon Alfa-2b (PEG Conjugate),Interleukin-2, Interleukin-11, Intron A® (interferon alfa-2b), Iressa®,Irinotecan, Isotretinoin, Ixabepilone, Ixempra™, Kidrolase, Lanacort®,Lapatinib, L-asparaginase, LCR, Lenalidomide, Letrozole, Leucovorin,Leukeran, Leukine™, Leuprolide, Leurocristine, Leustatin™, LiposomalAra-C, Liquid Pred®, Lomustine, L-PAM, L-Sarcolysin, Lupron®, LupronDepot®, Matulane®, Maxidex, Mechlorethamine, MechlorethamineHydrochloride, Medralone®, Medrol®, Megace®, Megestrol, MegestrolAcetate, Melphalan, Mercaptopurine, Mesna, Mesnex™, Methotrexate,Methotrexate Sodium, Methylprednisolone, Meticorten®, Mitomycin,Mitomycin-C, Mitoxantrone, M-Prednisol®, MTC, MTX, Mustargen®, Mustine,Mutamycin®, Myleran®, Mylocel™, Mylotarg®, Navelbine®, Nelarabine,Neosar®, Neulasta™, Neumega®, Neupogen®, Nexavar®, Nilandron®,Nilutamide, Nipent®, Nitrogen Mustard, Novaldex®, Novantrone®,Octreotide, Octreotide acetate, Oncospar®, Oncovin®, Ontak®, Onxal™,Oprevelkin, Orapred®, Orasone®, Oxaliplatin, Paclitaxel, PaclitaxelProtein-bound, Pamidronate, Panitumumab, Panretin®, Paraplatin®,Pediapred®, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON™,PEG-L-asparaginase, PEMETREXED, Pentostatin, Phenylalanine Mustard,Platinol®, Platinol-AQ®, Prednisolone, Prednisone, Prelone®,Procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 with CarmustineImplant Purinethol®, Raloxifene, Revlimid®, Rheumatrex®, Rituxan®,Rituximab, Roferon-A® (Interferon Alfa-2a), Rubex®, Rubidomycinhydrochloride, Sandostatin® Sandostatin LAR®, Sargramostim,Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™, STI-571, Streptozocin,SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretin®, Taxol®,Taxotere®, Temodar0, Temozolomide, Temsirolimus, Teniposide, TESPA,Thalidomide, Thalomid®, TheraCys®, Thioguanine, Thioguanine Tabloid®,Thiophosphoamide, Thioplex®, Thiotepa, TICE®, Toposar®, Topotecan,Toremifene, Torisel®, Tositumomab, Trastuzumab, Treanda®, Tretinoin,Trexall™, Trisenox®, TSPA, TYKERB®, VCR, Vectibix™, Velban®, Velcade®,VePesid®, Vesanoid®, Viadur™, Vidaza®, Vinblastine, Vinblastine Sulfate,Vincasar Pfs®, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB,VM-26, Vorinostat, VP-16, Vumon®, Xeloda®, Zanosar®, Zevalin™,Zinecard®, Zoladex®, Zoledronic acid, Zolinza, Zometa®.

Routes of Administration

Antibodies, antigen binding fragments, polypeptides and othertherapeutic agents, medicaments and pharmaceutical compositionsaccording to aspects of the present invention may be formulated foradministration by a number of routes, including but not limited to,parenteral, intravenous, intra-arterial, intramuscular, subcutaneous,intradermal, intratumoral and oral. Antibodies, antigen bindingfragments, polypeptides and other therapeutic agents, may be formulatedin fluid or solid form. Fluid formulations may be formulated foradministration by injection to a selected region of the human or animalbody.

Dosage Regime

Multiple doses of the antibody, antigen binding fragment or polypeptidemay be provided. One or more, or each, of the doses may be accompaniedby simultaneous or sequential administration of another therapeuticagent.

Multiple doses may be separated by a predetermined time interval, whichmay be selected to be one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or31 days, or 1, 2, 3, 4, 5, or 6 months. By way of example, doses may begiven once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).

Kits

In some aspects of the present invention a kit of parts is provided. Insome embodiments the kit may have at least one container having apredetermined quantity of the antibody, antigen binding fragment orpolypeptide. The kit may provide the antibody, antigen binding fragmentor polypeptide in the form of a medicament or pharmaceuticalcomposition, and may be provided together with instructions foradministration to a patient in order to treat a specified disease orcondition. The antibody, antigen binding fragment or polypeptide may beformulated so as to be suitable for injection or infusion to a tumor orto the blood.

In some embodiments the kit may further comprise at least one containerhaving a predetermined quantity of another therapeutic agent (e.g.anti-infective agent or chemotherapy agent). In such embodiments, thekit may also comprise a second medicament or pharmaceutical compositionsuch that the two medicaments or pharmaceutical compositions may beadministered simultaneously or separately such that they provide acombined treatment for the specific disease or condition. Thetherapeutic agent may also be formulated so as to be suitable forinjection or infusion to a tumor or to the blood.

Subjects

The subject to be treated may be any animal or human. The subject ispreferably mammalian, more preferably human. The subject may be anon-human mammal, but is more preferably human. The subject may be maleor female. The subject may be a patient.

A subject may have been diagnosed with a disease or condition requiringtreatment, or be suspected of having such a disease or condition.

Protein Expression

Molecular biology techniques suitable for producing polypeptidesaccording to the invention in cells are well known in the art, such asthose set out in Sambrook et al., Molecular Cloning: A LaboratoryManual, New York: Cold Spring Harbor Press, 1989

The polypeptide may be expressed from a nucleotide sequence. Thenucleotide sequence may be contained in a vector present in a cell, ormay be incorporated into the genome of the cell.

A “vector” as used herein is an oligonucleotide molecule (DNA or RNA)used as a vehicle to transfer exogenous genetic material into a cell.The vector may be an expression vector for expression of the geneticmaterial in the cell. Such vectors may include a promoter sequenceoperably linked to the nucleotide sequence encoding the gene sequence tobe expressed. A vector may also include a termination codon andexpression enhancers. Any suitable vectors, promoters, enhancers andtermination codons known in the art may be used to express polypeptidesfrom a vector according to the invention. Suitable vectors includeplasmids, binary vectors, viral vectors and artificial chromosomes (e.g.yeast artificial chromosomes).

In this specification the term “operably linked” may include thesituation where a selected nucleotide sequence and regulatory nucleotidesequence (e.g. promoter and/or enhancer) are covalently linked in such away as to place the expression of the nucleotide sequence under theinfluence or control of the regulatory sequence (thereby forming anexpression cassette). Thus a regulatory sequence is operably linked tothe selected nucleotide sequence if the regulatory sequence is capableof effecting transcription of the nucleotide sequence. Whereappropriate, the resulting transcript may then be translated into adesired protein or polypeptide.

Any cell suitable for the expression of polypeptides may be used forproducing peptides according to the invention. The cell may be aprokaryote or eukaryote. Suitable prokaryotic cells include E.coli.Examples of eukaryotic cells include a yeast cell, a plant cell, insectcell or a mammalian cell. In some cases the cell is not a prokaryoticcell because some prokaryotic cells do not allow for the samepost-translational modifications as eukaryotes. In addition, very highexpression levels are possible in eukaryotes and proteins can be easierto purify from eukaryotes using appropriate tags. Specific plasmids mayalso be utilised which enhance secretion of the protein into the media.

Methods of producing a polypeptide of interest may involve culture orfermentation of a cell modified to express the polypeptide. The cultureor fermentation may be performed in a bioreactor provided with anappropriate supply of nutrients, air/oxygen and/or growth factors.Secreted proteins can be collected by partitioning culturemedia/fermentation broth from the cells, extracting the protein content,and separating individual proteins to isolate secreted polypeptide.Culture, fermentation and separation techniques are well known to thoseof skill in the art.

Bioreactors include one or more vessels in which cells may be cultured.Culture in the bioreactor may occur continuously, with a continuous flowof reactants into, and a continuous flow of cultured cells from, thereactor. Alternatively, the culture may occur in batches. The bioreactormonitors and controls environmental conditions such as pH, oxygen, flowrates into and out of, and agitation within the vessel such that optimumconditions are provided for the cells being cultured.

Following culture of cells that express the polypeptide of interest,that polypeptide is preferably isolated. Any suitable method forseparating polypeptides/proteins from cell culture known in the art maybe used. In order to isolate a polypeptide/protein of interest from aculture, it may be necessary to first separate the cultured cells frommedia containing the polypeptide/protein of interest. If thepolypeptide/protein of interest is secreted from the cells, the cellsmay be separated from the culture media that contains the secretedpolypeptide/protein by centrifugation. If the polypeptide/protein ofinterest collects within the cell, it will be necessary to disrupt thecells prior to centrifugation, for example using sonification, rapidfreeze-thaw or osmotic lysis. Centrifugation will produce a pelletcontaining the cultured cells, or cell debris of the cultured cells, anda supernatant containing culture medium and the polypeptide/protein ofinterest.

It may then be desirable to isolate the polypeptide/protein of interestfrom the supernatant or culture medium, which may contain other proteinand non-protein components. A common approach to separatingpolypeptide/protein components from a supernatant or culture medium isby precipitation. Polypeptides/proteins of different solubility areprecipitated at different concentrations of precipitating agent such asammonium sulfate. For example, at low concentrations of precipitatingagent, water soluble proteins are extracted. Thus, by adding increasingconcentrations of precipitating agent, proteins of different solubilitymay be distinguished. Dialysis may be subsequently used to removeammonium sulfate from the separated proteins.

Other methods for distinguishing different polypeptides/proteins areknown in the art, for example ion exchange chromatography and sizechromatography. These may be used as an alternative to precipitation, ormay be performed subsequently to precipitation.

Once the polypeptide/protein of interest has been isolated from cultureit may be necessary to concentrate the protein. A number of methods forconcentrating a protein of interest are known in the art, such asultrafiltration or lyophilisation.

Sequence Identity

Alignment for purposes of determining percent amino acid or nucleotidesequence identity can be achieved in various ways known to a person ofskill in the art, for instance, using publicly available computersoftware such as ClustalW 1.82. T-coffee or Megalign (DNASTAR) software.When using such software, the default parameters, e.g. for gap penaltyand extension penalty, are preferably used. The default parameters ofClustalW 1.82 are: Protein Gap Open Penalty=10.0, Protein Gap ExtensionPenalty=0.2, Protein matrix=Gonnet, Protein/DNA ENDGAP=−1, Protein/DNAGAPDIST=4.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Ranges may be expressedherein as from “about” one particular value, and/or to “about” anotherparticular value. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by theuse of the antecedent “about,” it will be understood that the particularvalue forms another embodiment.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments and experiments illustrating the principles of the inventionwill now be discussed with reference to the accompanying figures inwhich:

FIG. 1. Light chain variable domain sequences for anti-PD-L1 antibodyclones A1, C2, C4, H12 and H12_GL. CDRs are underlined and shownseparately.

FIG. 2. Heavy chain variable domain sequences for anti-PD-L1 antibodyclones A1, C2, C4, H12 and H12_GL. CDRs are underlined and shownseparately.

FIG. 3. Table showing light chain and heavy chain CDR sequences foranti-PD-L1 antibody clones A1, C2, C4, H12 and H12_GL.

FIG. 4. Nucleotide and encoded amino acid sequences of heavy and lightchain variable domain sequences for anti-PD-L1 antibody clones A1, C2,C4, H12 and H12_GL.

FIG. 5. Bar Chart showing binding of anti-PD-L1, human Fc conjugatedantibodies A1, C2, C4 and H12 and control antibodies A3 (which binds tohuman but not murine PD-L1) and E3 (which does not bind to human ormurine PD-L1).

FIG. 6. Chart showing inhibition of human PD-1/human PD-L1 interactionby human Fc conjugated antibodies A1, C2, C4 and H12, as determined byELISA.

FIG. 7. Chart showing inhibition of murine PD-1/murine PD-L1 interactionby human Fc conjugated antibodies A1, C2, C4 and H12, as determined byELISA.

FIG. 8. Chart showing inhibition of murine PD-1/murine PD-L1 interactionby H12 (IgG1) and commercial anti-mouse PD-L1 antibody.

FIG. 9. Bar Chart showing IFNγ secretion of exhausted T cells inresponse to antibodies H12, nivolumab (commercial anti-PD-1 antibody),and isotype and no antibody control.

FIG. 10. Bar Chart showing proliferation of exhausted T cells inresponse to antibodies anti-PD-L1 H12, nivolumab, and isotype and noantibody control.

FIG. 11. Sensorgram showing affinity of anti-PD-L1 H12 for human PD-L1as determined by surface plasmon resonance (SPR). KD=3.13×10⁻¹⁰ M.

FIG. 12. Sensorgram showing affinity of anti-PD-L1 H12 for murine PD-L1as determined by surface plasmon resonance (SPR). KD=3.04×10⁻⁹ M.

FIG. 13. Bar Charts showing IFNγ secretion (A) of dissociated lung tumortissue and

(B) dissociated lung tumor tissue co-cultured with allogenic dendriticcells (DC) in a mixed lymphocyte reaction (MLR), in response tonivolumab, labrolizumab (both commercial anti-PD-1 antibodies), antibodyH12, and isotype and no antibody controls.

FIG. 14. Bar Chart showing IFNγ secretion after culture of PBMCs withInfluenza in the presence of the nivolumab, labrolizumab, antibodyH12-IgG₄, antibody H12-IgG₁, and isotype and no antibody controls.

FIG. 15. Bar Chart showing ELISA with H12 on different antigens: human(hu) PD-1, PD-L1, PD-L2, TIM-3, LAG-3, CTL-A4, BLTA, ICOS and CD28, andcynomolgus (cy) and mouse (mo) PD-L1.

FIG. 16. Graphs showing inhibition of tumour growth by antibody H12 in(A) a mouse colon cancer model using CT26 cell line and (B) a mousemelanoma model using B16F10 cell line.

FIG. 17. Graph showing efficiency of blocking of interaction betweenPD-1 and PD-L1 by antibodies H12-IgG₁, H12-IgG₄, H12_GL-IgG₄, andH12_GL-IgG₄, as determined by blocking assay using HEK293-6E cellstransfected with human PD-L1.

FIG. 18. Graphs showing in vivo efficiency of anti-PD-L1 antibody H12 tocontrol tumour growth in a mouse model. MC38 cells were inoculated intomice, and five doses of 200 μg per animal of the indicated antibody weregiven intraperitoneally starting at day 8. (A) and (B) show the resultsof tumour growth (mean±SEM) for two independent experiments.

FIG. 19. Graphs showing binding of antibodies H12, C4 and atezolizumabto human PD-L1, and avidity of binding. (A) Binding of H12, C4,atezolizumab and isotype control antibodies to human PD-L1 (mean±SD ofduplicates). (B) Avidity of binding of H12, C4, atezolizumab and isotypecontrol antibodies to human PD-L1 (mean±SD of duplicates).

FIG. 20. Graph showing binding of H12 IgG1, C4 IgG1, atezolizumab andDEN IgG1 antibodies to MDA-MB-231 human breast cancer cells. Meanfluorescence index (MFI) is shown (mean±SD of duplicates).

FIG. 21. Graph showing binding of PD-L1 to PD-1 in the presence of H12,C4, atezolizumab and isotype control antibodies. The graph shows abilityof antibodies to block binding of PD-L1 to its receptor PD-1 (mean±SD ofduplicates).

EXAMPLES

The inventors describe in the following Examples the identification ofnucleotide and amino-acid sequences of isolated antibodies, or theantigen-binding portions thereof, that specifically bind human andmurine PD-L1, block the PD-1 pathway and restore exhausted T cellactivity.

Example 1 Isolation of Anti-Human PD-L1 Antibodies

Anti-PD-L1 antibodies were isolated from a human antibody phage displaylibrary via in vitro selection.

Streptavidin-magnetic beads were coated with biotinylated human PD-L1and used to fish-out anti-PD-L1-specific phages using magnetic sorting.Some steps to remove potential anti-biotin antibodies were added in theselection process.

Specific Fab antibodies were originally identified by ELISA withhuman-PD-L1 as the antigen. Four clones were retained for furthercharacterisation based on their slow dissociation from human PD-L1:clones A1, C2, C4 and H12. A first clonality screening was performed byDNA fingerprinting; clonality was then confirmed by sequencing.

Example 2 Binding to Human and Murine PD-L1

Human and murine PD-L1 were coupled to human Fc and used as antigenscoated on ELISA plates for investigation of antibody binding.

Briefly, ELISA plates were coated with human or murine PD-L1-Fc incarbonate buffer, plates were then blocked with a solution of casein andafter extensive washes in PBS Tween-20, anti-PD-L1 antibodies in Fabformat were added into the ELISA wells in the presence of 7% milk inPBS. After 90 minutes at room temperature under agitation and extensivewashes, a goat anti-human Fab antibody coupled to HRP was added. Onehour later, plates were washed and TMB substrate added. The reaction wasstopped with 1M HCl and optical density measured at 450 nm with areference at 670 nm.

The results are shown in FIG. 5. A1, C2, C4 and H12 antibodies werefound to be capable of recognising both human and murine PD-L1 (solidand hatched bars, respectively), and not the coupled Fc part (openbars). Clone A3, which recognises human but not murine PD-L1, and cloneE3 which does not recognise either human or murine PD-L1, were includedas controls.

Antibody clones A1, C2, C4 and H12 were each found to be cross reactivefor human PD-L1 and murine PD-L1. H12 demonstrated similar binding tohuman PD-L1 and murine PD-L1.

Example 3 Blocking the PD-1/PD-L1 Interaction In Vitro

Anti-PD-L1 antibodies were investigated for their ability to blockbinding of PD-L1 to PD-1 by ELISA assay using PD-1 coupled to human Fcas an antigen. Biotinylated human or murine PD-L1 was pre-incubated inthe presence of A1, C4, C2 or H12 Fab prior to addition onto PD-1.Binding of PD-L1 to PD-1 was determined using streptavidin-HRP/TMBsubstrate. The results of these investigations are shown in FIGS. 6 and7.

Antibodies A1, C2, C4 and H12 expressed as Fabs were all found toeffectively inhibit the binding of human PD-L1 to human PD-1 in adose-dependent manner (FIG. 6). Clones A1, C2 and C4 were found toinhibit this interaction with similar efficiency, whilst H12 was foundto be slightly more effective than A1, C2 and C4 (FIG. 6).

Antibody A1, C2, C4 and H12 expressed as Fabs were also found to be ableto block the binding of murine PD-L1 to murine PD-1 (FIG. 7). Whilstantibodies A1, C2 and C4 were found to disrupt interaction betweenmurine PD-L1 and murine PD-1 at concentrations higher than theconcentration required to block binding of human PD-L1 to human PD-1,H12 was found to be capable of neutralising binding at concentrationssimilar to those required to block interaction between human PD-L1 andhuman PD-1 (FIG. 7).

Inhibition of binding between murine PD-L1 and murine PD-1 was alsoinvestigated by antibody H12 expressed in the IgG1 format, and comparedto inhibition by a commercially available anti-murine PD-L1 IgG antibody[10F.9G2 (BioLegend, Inc, San Diego, Calif., USA)]. The results areshown in FIG. 8. Antibody H12 was found to be significantly moreeffective at inhibiting interaction between murine PD-L1 and murine PD-1at lower antibody concentrations as compared to the commerciallyavailable anti-murine PD-L1 IgG antibody.

Example 4 Restoration of Exhausted T Cell Activity

Ability of anti-PD-L1 antibody H12 to restore exhausted T cell activitywas investigated.

Briefly, T cells were isolated from a healthy donor and cultured for 7days with monocyte-derived dendritic cells obtained from another donor(50,000 T cells/5,000 DCs), in an allogeneic reaction. The T cellsunderwent two rounds of stimulation to achieve exhaustion. The exhaustedT cells were cultured in the presence of antibody H12 or the anti-PD-1antibody Nivolumab in the second round of stimulation for 5 days, priorto measurement of IFN-γ in the supernatants and quantification ofproliferation using tritiated thymidine.

The results are shown in FIGS. 9 and 10. Antibody H12 was found tosuppress T cell exhaustion, restoring both secretion of IFN-y (FIG. 9)and proliferation (FIG. 10) of T cells. Proliferation and IFN-γsecretion were restored to a similar level by treatment with H12 at 1000ng/ml as with treatment with Nivolumab at 1000 ng/ml. Notably,proliferation and IFN-γ secretion was higher for T cells treated withH12 antibody than Nivolumab at 200 ng/ml and 40 ng/ml concentrations.

Example 5 Antibody Affinity for PD-L1

Affinity for antibody H12 for human PD-L1 and murine PD-L1 wasinvestigated by Surface Plasmon Resonance (SPR) analysis. Briefly, humanor mouse PD-L1 coupled to Fc was immobilised on a sensor chip compatiblewith the Proteon XPR36 Bioanalyser (BioRad). Crude H12 Fab extract wasthen flown onto the chip and the association/dissociation was recordedand analysed and the affinity (K_(D)) was calculated.

The results are shown in FIGS. 11 and 12. H12 was found to have a highaffinity for human PD-L1 of K_(D)=0.313 nM, and for murine PD-L1 ofK_(D)=3.04 nM.

Example 6 Use of Anti-PD-L1 Antibodies to Treat Tumours: Ex VivoActivation of Tumor Infiltrating Lymphocytes

Lung tumour samples were obtained from the National Cancer CentreSingapore after approval. Samples were dissociated using a human tumourdissociation kit and a tissue dissociator device.

The tumour dissociated mixture was cultured with anti-PDL-1 IgG, cloneH12 for 7 days prior to measurement of IFN-γ in the supernatant byELISA. Nivolumab and lambrolizumab were used as positive controls in theassay (both are anti-PD-1 antibodies), an isotype antibody was used as anegative control.

FIG. 13A shows secretion of IFN-γ by tumour infiltrating lymphocytesafter 7 days of culture in the presence of the antibodies. H12re-activated lymphocytes to secrete IFN-γ in a dose-dependent manner.

Another fraction of the dissociated mixture was co-cultured withallogeneic dendritic cells (DC) to initiate a mixed lymphocyte reaction(MLR). Cells were first cultured for 7 days without antibodies and thenfor 7 days in the presence of H12 or control antibodies. After these 2rounds, IFN-γ was assayed in supernatants by ELISA.

FIG. 13B shows secretion of IFN-γ after the MLR in the presence of theantibodies. H12 restored the ability of tumour lymphocytes to secreteIFN-γ in a dose-dependent manner.

Example 7 Use of Anti-PD-L1 Antibodies to Treat Infections: AutolociousActivation of T Cells in the Presence of Influenza

Blood was collected from Influenza-positive donors. Monocyte-derived DCswere infected with influenza virus A/PR/8/34 (H1N1). Infected DCs weremixed to PBMCs from the same donor for a first round of culture of 5days. Cells were then cultured for a second round of 5 days in thepresence of H12 or control antibodies. After these 2 rounds, most of thecells in culture are Influenza-specific T cells. At the end of 2 roundsof culture, IFN-γ was assayed in supernatants by ELISA. In this assay,H12 was tested either as an IgG, antibody or as an IgG₄ antibody.

FIG. 14 shows secreted IFN-γ after culture of PBMCs with Influenza inthe presence of the antibodies. Both H12-IgG₁ and H12-IgG₄ were able torestore the capacity of lymphocytes to secrete IFN-γ upon viralstimulation in a dose-dependent manner.

Example 8 Specificity/Cross-Reactivity of H12

Recognition of various members of the CD28 family by H12 was tested byELISA.

FIG. 15 shows the ELISA with H12 on different antigens: human (hu) PD-1,PD-L1, PD-L2, TIM-3, LAG-3, CTL-A4, BLTA, ICOS and CD28, and cynomolgus(cy) and mouse (mo) PD-L1. H12 displayed specificity for the PD-L1molecule, and cross-reactivity among species.

Example 9 Preliminary In Vivo Efficacy of H12 Antibody

H12 was tested in 2 tumour models: a colon cancer model using CT26 cellline and a melanoma model using B16F10 cell line.

Balb/C or C57BL/6 mice were inoculated in the left flank with 0.5×10⁶CT26 or 0.2×10⁶ B16F10 tumour cells, respectively, at day 0, andinjected intraperitoneally with 250 μg of H12 or isotype IgG1 at days 7,11, 14, 18 and 21. Tumour growth was then analysed, size was measuredwith a caliper and volume calculated as follows: V=l²×L/2 (withl=shorter side and L=longer side).

FIGS. 16A and 16B show tumour growth after tumour inoculation in bothmodels. H12 inhibited tumour growth in both models.

Example 10 Data with Engineered H12 Antibody

H12 clone was engineered in order to revert its framework to agermline-like framework; the heavy chain of the new clone H12_GL wasslightly modified, the light chain remained the same as for H12 originalclone.

H12_GL was tested in a blocking assay. Briefly HEK 293 cells weretransfected with the pcDNA3.1 plasmid expressing human PD-L1 proteinusing 293tfectin (Invitrogen), according to manufacturer's protocol.PD-L1 expression was confirmed with commercial anti-mouse or anti-humanPD-L1 conjugated to PECy7 (Biolegend). 50 to 100 nM of recombinant humanPD-1 labelled with Phycoerythrin (PE) was used to bind the PD-L1expressed on the transfected cells. Serially diluted antibodies(anti-PD-L1 H12, H12_GL or isotype control) were mixed with the humanPD-1-PE for 30 minutes before being added to the PD-L1 transfectedcells. Cells were washed 3 times with PBS and all data were collected ona BD FACSCanto II (BD Bioscience) and analysed on BD FACSDiva software(BD Bioscience).

FIG. 17 shows that H12_GL blocked PD-1/PD-L1 interactions as efficientlyas H12.

Example 11 In Vivo Efficacy of Anti-PD-L1 Antibody H12: Control ofTumour Growth in Mouse Model

Because H12 was shown to cross-react with mouse PD-L1, this antibody wasevaluated for ability to control tumour growth in a mouse model usingcolon carcinoma MC38 cells.

Two million MC38 cells were inoculated to mice subcutaneously at day 0,and five doses of 200 μg per animal of the anti-PD-L1 H12 IgG₁ orisotype control antibody were injected intra-peritoneally, starting atday 8. Tumour size was measured throughout the experiment.

The results for two independent experiments are shown in FIGS. 18A and18B. The graphs show tumour growth (mean±SEM) in the MC38 tumour growthmodel in the presence of anti-PD-L1 antibody H12 or negative isotypecontrol. In both experiments, H12 was able to control tumour growth.

Example 12 Binding of Anti-PD-L1 mAbs to the Target and Comparison withAtezolizumab

Binding of anti-PD-L1 clones H12 and C4 to human PD-L1 was compared tothat of atezolizumab, a commercially available anti-PD-L1 IgG1 antibody.

Antibodies were coated to a plate and various concentrations ofbiotinylated PD-L1 were added. Binding was measured by ELISA. Antibodieswere coated onto maxisorp plates at 2 μg/mL in coating buffer andincubated overnight at 4° C. The next day, plates were washed with washbuffer (PBS+0.05% Tween-20), and blocker with casein for 1 hour, at roomtemperature. Plates were then washed again using wash buffer. Variousconcentrations of biotinylated human PD-L1 were then added to theplates, and the plates were then incubated at room temperature for 1hour. Plates were then washed again using wash buffer. Streptavidin-HRPwas then added and incubated for 1 hour at room temperature to detectbiotinylated human PD-L1 bound to the different antibodies. Plates werethen washed again using wash buffer. Finally, TMB was added to developthe ELISA; TMB conversion by HRP was stopped using 1M H—Cl.

Avidity was also assessed by ELISA, with PD-L1 antigen bound onto aplate at single concentration, and addition of various concentrations ofantibodies. A secondary antibody was used to detect anti-PD-L1 antibodycomplexed to the antigen. Neutravidin was coated onto maxisorp plates at2 μg/mL in coating buffer and incubated overnight at 4° C. The next day,plates were washed with wash buffer (PBS+0.05% Tween-20), and blockerwith casein for 1 hour, at room temperature. Plates were then washedagain using wash buffer. Biotinylated human PD-L1 was then added to theplates at 0.2 μg/mL and incubated for 1 hour at room temperature. Plateswere then washed again using wash buffer. The different antibodies werethen added at various concentrations, and incubated for 1 hour at roomtemperature. Plates were then washed again using wash buffer. Anti-humanFc-HRP was then added and incubated for 1 hour at room temperature.Plates were then washed again using wash buffer. Finally, TMB was addedto develop the ELISA; TMB conversion by HRP was stopped using 1M H—Cl.

FIG. 19A presents the binding of the antibodies (and isotype control) toPD-L1 (mean±SD of duplicates). H12 shows a similar binding curve forPD-L1 as atezolizumab. Affinity of H12 for PD-L1 is higher than affinityof atezolizumab for PD-L1.

FIG. 19B presents the avidity of the antibodies for their target(mean±SD of duplicates). H12 shows a high avidity to PD-L1, similar toatezolizumab, while C4 shows a medium avidity. Avidity of H12 for PD-L1is higher than avidity of atezolizumab for PD-L1.

Binding to PD-L1 was also tested by investigating binding to aMDA-MB-231 human breast cancer cell line that constitutively expressesPD-L1 at the cell membrane. Binding of the antibodies was measured byflow cytometry.

FIG. 20 presents the mean fluorescence index (MFI) for differentconcentrations of antibodies (mean±SD of duplicates). The anti-PD-L1antibodies H12 IgG1 and C4 IgG1 bind efficiently to cells expressingPD-L1.

Example 13 In Vitro Activity of Anti-PD-L1 Antibodies: Blocking of PD-L1Binding to PD-1

The ability of the antibodies to block the binding of PD-L1 to itsreceptor PD-1 was assessed by ELISA. Briefly, the antibodies werepre-incubated with PD-L1 and then added onto ELISA plates onto whichPD-1 was coated. After washing, the presence of PD-1-bound PD-L1 wasdetected with an antibody.

FIG. 21 presents the results of experiments investigating binding ofPD-L1 to PD-1 in the presence or absence of anti-PD-L1 antibodies (orisotype control) (mean±SD of duplicates). The anti-PD-L1 antibodies H12and C4 are able to block the binding to PD-L1 to its receptor PD-1. BothH12 and C4 were able to block interaction between PD-L1 and PD-1 withgreater efficiency than atezolizumab.

1-31. (canceled)
 32. An antibody, or antigen binding fragment, which iscapable of binding to PD-L1, optionally isolated, having at least onelight chain variable region incorporating the following CDRs:(SEQ ID NO: 18) LC-CDR1: TGSSSNIGAGYDVH (SEQ ID NO: 19) LC-CDR2: GNSNRPS(SEQ ID NO: 20) LC-CDR3: QSYDSSLSGSYVV;

and having at least one heavy chain variable region incorporating thefollowing CDRs: (SEQ ID NO: 21) HC-CDR1: SYAIS (SEQ ID NO: 22)HC-CDR2: RIIPILGIANYAQKFQG (SEQ ID NO: 25) HC-CDR3: SGHGYSYGAFDY.


33. The antibody, or antigen binding fragment, according to claim 32,that specifically binds to human or murine PD-L1.
 34. The antibody, orantigen binding fragment, according to claim 32, that inhibitsinteraction between human PD-1 and human PD-L1, or inhibits interactionbetween murine PD-1 and murine PD-L1.
 35. The antibody, or antigenbinding fragment, of claim 32, wherein the antibody is effective torestore T-cell function in T-cells exhibiting T-cell exhaustion orT-cell anergy.
 36. The antibody or antigen binding fragment of claim 32,comprising a heavy chain variable region sequence and a light chainvariable region sequence, wherein: the heavy chain variable regionsequence has at least 70% sequence identity to the heavy chain variableregion sequence of SEQ ID NO:8 or 35 (FIG. 2), and the light chainvariable region sequence has at least 70% sequence identity to the lightchain variable region sequence of SEQ ID NO:4 (FIG. 1).
 37. The antibodyor antigen binding fragment of claim 32, which is a bispecific antibodyor a bispecific antigen binding fragment comprising (i) an antigenbinding fragment according to claim 3, and (ii) an antigen bindingfragment which is capable of binding to a target protein other thanPD-L1.
 38. The antibody, or antigen binding fragment, of claim 37,wherein the antigen binding fragment which is capable of binding to atarget protein other than PD-L1 is capable of binding to one of CD27,CD28, ICOS, CD40, CD122, OX40, 4-1BB, GITR, LAG-3, B7-H3, B7-H4, BTLA,CTLA-4, A2AR, VISTA, TIM-3, PD-1, KIR, HER-2, HER-3, EGFR, EpCAM, CD30,CD33, CD38, CD20, CD24, CD90, CD15, CD52, CA-125, CD34, CA-15-3,CA-19-9, CEA, CD99, CD117, CD31, CD44, CD123, CD133, ABCB5 and CD45. 39.A method of treating a T-cell dysfunctional disorder, cancer or aninfectious disease, comprising administering an antibody or antigenbinding fragment to a patient suffering from a T-cell dysfunctionaldisorder, cancer or an infectious disease, wherein antibody, or antigenbinding fragment, is capable of binding to PD-L1, and comprises: atleast one light chain variable region incorporating the following CDRs:(SEQ ID NO: 18) LC-CDR1: TGSSSNIGAGYDVH (SEQ ID NO: 19) LC-CDR2: GNSNRPS(SEQ ID NO: 20) LC-CDR3: QSYDSSLSGSYVV;

and at least one heavy chain variable region incorporating the followingCDRs: (SEQ ID NO: 21) HC-CDR1: SYAIS (SEQ ID NO: 22)HC-CDR2: RIIPILGIANYAQKFQG (SEQ ID NO: 25) HC-CDR3: SGHGYSYGAFDY.


40. A method for expanding a population of T cells, wherein T cells arecontacted in vitro or ex vivo with an antibody or antigen bindingfragment, wherein antibody, or antigen binding fragment, is capable ofbinding to PD-L1, and comprises: at least one light chain variableregion incorporating the following CDRs: (SEQ ID NO: 18)LC-CDR1: TGSSSNIGAGYDVH (SEQ ID NO: 19) LC-CDR2: GNSNRPS (SEQ ID NO: 20)LC-CDR3: QSYDSSLSGSYVV;

and at least one heavy chain variable region incorporating the followingCDRs: (SEQ ID NO: 21) HC-CDR1: SYAIS (SEQ ID NO: 22)HC-CDR2: RIIPILGIANYAQKFQG (SEQ ID NO: 25) HC-CDR3: SGHGYSYGAFDY.


41. The antibody or antigen binding fragment of claim 32, comprising aheavy chain variable region sequence and a light chain variable regionsequence, wherein: the heavy chain variable region sequence has at least85% sequence identity to the heavy chain variable region sequence of SEQID NO:8 (FIG. 2), and the light chain variable region sequence has atleast 85% sequence identity to the light chain variable region sequenceof SEQ ID NO:4 (FIG. 1).
 42. The antibody or antigen binding fragment ofclaim 32, comprising a heavy chain variable region sequence and a lightchain variable region sequence, wherein: the heavy chain variable regionsequence has at least 85% sequence identity to the heavy chain variableregion sequence of SEQ ID NO:35 (FIG. 2), and the light chain variableregion sequence has at least 85% sequence identity to the light chainvariable region sequence of SEQ ID NO:4 (FIG. 1).
 43. The antigenbinding fragment of claim 32, wherein the antigen binding fragment is aFab fragment or scFv fragment.
 44. The antibody of claim 32, wherein theantibody comprises a human constant region selected from IgG1, IgG2,IgG3 and IgG4.
 45. The method according to claim 39, wherein the methodis a method of treating cancer, and comprises administering the antibodyor antigen binding fragment to a patient suffering from cancer.
 46. Themethod according to claim 45, wherein the cancer is a cancer of a tissueselected from the group consisting of lung, colon, skin, breast, liver,stomach, nasopharynx, oral cavity, oesophagus, larynx, salivary gland,tongue, tonsil, trachea, kidney, bladder, and blood.
 47. The methodaccording to claim 45, wherein the cancer is selected from the groupconsisting of lung cancer, non-small cell lung cancer (NSCLC), coloncancer, colon carcinoma, colorectal carcinoma, skin cancer, melanoma,metastatic melanoma, breast cancer, liver cancer, hepatoma, stomachcancer, head and neck cancer, nasopharyngeal cancer, oral cavity cancer,oesophageal cancer, laryngeal cancer, salivary gland cancer, tonguecancer, tonsil cancer, tracheal cancer, renal cancer, bladder cancer,haematologic cancer, lymphoma and Hodgkin's lymphoma.